Methods, kits, and compositions for stem cell self-renewal

ABSTRACT

The present invention relates to methods and kits for expanding a stem cell population. More particularly, the invention relates, inter alia, to methods, kits, and compositions for expanding a stem cell population, particularly a hematopoietic stem cell population.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationSerial No. PCT/US2008/005230, filed Apr. 23, 2008, which claims benefitto U.S. Provisional Patent Application Ser. No. 60/926,065, filed Apr.23, 2007 and U.S. Provisional Patent Application Ser. No. 61/066,693,filed Feb. 22, 2008. The entire contents of the above-mentionedapplications are hereby incorporated by reference as if recited in fullherein.

FIELD OF THE INVENTION

The present invention relates to methods, kits, and compositions forexpanding a stem cell population, particularly an hematopoietic stemcell population.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells (HSCs) are clonogenic cells, which possess theproperties of both self-renewal (expansion) and multilineage potentialgiving rise to all types of mature blood cells. HSCs are responsible forhematopoiesis and undergo proliferation and differentiation to producemature blood cells of various lineages while still maintaining theircapacity for self-renewal. The ability to self-renew maintains the HSCpopulation for the lifespan of an animal and also allows HSCs torepopulate the bone marrow of lethally irradiated congenic hosts.

Early HSC development displays a hierarchical arrangement, starting fromlong-term (LT-) HSCs, which have extensive self-renewal capability,followed by the expansion state, which corresponds to short-term (ST-)HSCs (having limited self-renewal ability) and proliferative multipotentprogenitors (MPPs) (having multipotent potential but no self-renewalcapability). MPP is also a stage of priming or preparation fordifferentiation. An MPP differentiates and commits to become either acommon lymphoid progenitor (CLP), which gives rise to all the lymphoidlineages, or a common myeloid progenitor (CMP), which produces all themyeloid lineages. During this process, the more primitive populationgives rise to a less primitive population of cells, which is unable togive rise to a more primitive population of cells. The intrinsic geneticprograms that control these processes including the multipotential,self-renewal, and activation (or transient amplification) of HSCs, andlineage commitment from MPP to CLP or CMP, remain largely unknown.

To sustain constant generation of blood cells for the lifetime of anindividual, HSCs located in bone marrow niches (Zhang, J. et al. Nature425, 836-841, 2003; Calvi, L. M. et al. Nature 425, 841-846, 2003; Kiel,M. J., et al. Cell 121, 1109-1121, 2005; Arai, F. et al. Cell 118,149-161, 2004) must achieve a balance between quiescence and activationso that immediate demands for hematopoiesis are fulfilled, whilelong-term stem cell maintenance is also assured. In adults, homeostasisbetween the quiescent and activated states of stem cells is important toprotect HSCs from losing their potential for self-renewal and, at thesame time, support ongoing tissue regeneration (Li, L. and Xie, T. Annu.Rev. Cell. Dev. Biol. 21, 605-631, 2005). Over-activation and expansionof stem cells risks both eventual depletion of the stem cell populationand a predisposition to tumorigenesis. Although some factors importantfor stem cell activation have been identified (Heissig, B. et al. Cell109, 625-637, 2002), the molecular events governing the transitionbetween quiescence and activation are poorly understood.

Phosphatase and tensin homolog (PTEN) functions as a negative regulatorof the PI3K/Akt pathway, which plays crucial roles in cellproliferation, survival, differentiation, and migration (Stiles, B. etal. Dev. Biol. 273, 175-184, 2004). The PTEN tumor suppressor iscommonly mutated in tumors, including those associated with lymphoidneoplasms, which feature deregulated hematopoiesis (Mutter, G. L. Am. J.Pathol. 158, 1895-1898, 2001; Suzuki, a. et al. Immunity 14, 523-534,2001). PTEN-deficiency has been associated with expansion of neural andembryonic stem cell populations (Groszer, M. et al. Science 294,2186-2189, 2001; Kimura, T. et al. Development 130, 1691-1700, 2003).But, the role of PTEN in stem cells and tumorigenesis and the recurrenceof tumors heretofore has been not understood.

PTEN functions as an antagonist of phosphatidyl inositol 3-kinase (PI3K)(Maehama T & Dixon J E. J Biol Chem. 273:13375-13378. 1998). The serinekinase Akt is downstream of the PI3K signal (Cross D A, Alessi D R,Cohen P et al. Nature 378:785-789 1995). PTEN has been shown to inhibitAkt and thereby inhibit the nuclear accumulation of β-catenin (Persad Set al. J Cell Biol. 153:1161-1174 2001).

Akt has a broad range of effects. Its major function is to provide asurvival signal and to block apoptosis, complementary to its regulationof β-catenin function. (Song, G. et al., J. Cell. Mol. Med., 9(1):59-71, 2005) Akt acts through a number of proteins, including mammaliantarget of rapamycin (mTOR), the Forkhead family of transcription factors(FoxO), BAD, caspase 9, murine double minute 2 (Mdm2).

Akt can directly and indirectly activate the serine/threonine kinasemTOR, which activates protein translation through a signaling cascade.(LoPiccolo, J., et al., Anti-Cancer Drugs, 18:861-874, 2007). Indirectactivation occurs through tuberous sclerosis complex-2 (TSC2), which,when in the unphosphorylated state, forms a complex with tuberoussclerosis complex-1 (TSC1, also known as hamartin). This complexpromotes the GTPase activity of Ras homolog enriched in brain (RHEB),which in turn, acts to down-regulate mTOR activity. Upon phosphorylationby Akt, however, the ability of the TSC1-TSC2 complex to promote RHEB'sGTPase activity is inhibited, and therefore, mTOR's activity ispromoted. (Cully, M. et al., Nat. Rev. Cancer, 6:184-192, 2006). mTORcan also form a complex with Rictor, and this complex can providepositive feedback on the Akt signaling cascade by phosphorylating andactivating Akt. (Sarbassov, D. D., et al., Science, 307: 1098-1101,2005).

Akt also regulates cell survival through transcriptional factors,including FoxO. Akt's phosphorylation of FoxO inhibits FoxO, resultingin inhibition of transcription of several proapoptotic genes, such asFas-L, IGFBP1 and Bim. (Datta, S. R., et al., Cell, 91:231-241, 1997;Nicholson, K. M., et al., Cell Signal, 14:381-395, 2002).

One of the down-stream targets of FoxO is p27 (Kip1), a potent inhibitorof cyclin E/cdk2 complexes. (Wu, H. et al., Oncogene, 22: 3113-3122,2003). FoxO factors induce expression of p27, which can bind to cyclinE/cdk2 complexes and inhibit their activity, resulting in a block incellular proliferation. (Burgering, B. M. T. & Medema, R. H., J.Leukocyte Biol., 73:689-701, 2003). In addition, Akt itself can alsodirectly phosphorylate p27 on T157, resulting in the redistribution ofp27 from the nucleus to the cytoplasm, away from cyclin E/cdk2complexes. (Id.) Phosphorylation of p27 on T198 was critical for thebinding of p27 to 14-3-3 proteins, and through this pathway, Akt maydirectly promote p27's degradation. (Fujita, N., et al., J. Biol. Chem.,277(32): 28706-28713, 2002).

Another one of the targets of Akt in promoting cell survival is BAD, amember of the Bcl-2 family of proteins. In the absence of Aktphosphorylation, BAD forms a complex with Bcl-2 or Bcl-X on themitochondrial membrane and inhibits the anti-apoptotic potential ofBcl-2 and Bcl-X. (Song, G. et al., J. Cell. Mol. Med., 9(1): 59-71,2005) Akt phosphorylates BAD on Serine 136, thus releasing BAD from theBcl-2/Bcl-X complex. (Song, G. et al., J. Cell. Mol. Med., 9(1): 59-71,2005; Datta, S. R., et al., Genes Dev., 13:2905-2927, 1999). Therefore,Akt suppresses BAD-mediated apoptosis and promotes cell survival.

Furthermore, by phosphorylation of pro-caspase-9 at Serine 196, Aktinhibits proteolytic processing of pro-caspase-9 to the active form,caspase-9, which is an initiator and an effecter of apoptosis (Cardoneet al., 1998, Science, 282: 1318-1320, Donepudi, M. & Grutter, M. G.,Biophys. Chem., 145-152, 2002).

Additionally, Akt regulates cell survival via the Mdm2/p53 pathway. Aktcan activate Mdm2 by direct phosphorylation, thereby inducing thenuclear import of Mdm2 or the up-regulation of Mdm2's ubiquitin ligaseactivity. (Mayo L. D., Donner D. B., 2001, Proc. Natl., Acad. Sci. USA98:11598-11603; Gottlieb T. M. et al, Ocogene, 21: 1299-1303, 2002).Mdm2 negatively regulates the p53 protein, which may induce cell deathin response to stresses (Oren M., Cell Death Differ., 10:431-442, 2003),by targeting p53 for ubiquitin-mediated proteolysis (Haupt, Y. et al.,1997, Nature 387: 296-299) or by binding to the transactivation domainof p53, thereby inhibiting p53-mediated gene regulation. (Momand, J. etal., Cell, 69: 1237-1245, 1992) One of the down-stream targets of p53 isthe p21 (CIP1/WAF1) gene. The p53 gene product binds to a site located2.4 kb upstream of the p21 coding sequence, and this binding siteconfers p53-dependent transcriptional regulation. (El-Deiry, W. S., etal., Cell, 75: 817-825, 1993) Thus, down-regulation of p53 alsodown-regulates the transcription of p21.

PTEN not only regulates p53 protein through antagonizing the Akt-Mdm2pathway, it can also directly regulate p53. First, PTEN can enhance p53transactivation in a phosphatase-independent manner (Tang, Y. & Eng C.,Cancer Research, 66: 736-742, 2006). Second, PTEN forms a complex withp300 in the nucleus and plays a role in maintenance of high p53acetylation, which is the activated form of p53. (Li A. et al.,Molecular Cell, 23 (4): 575-587, 2006). In turn, p53 may also activatethe transcription of PTEN. (Cully, M. et al., Nat. Rev. Cancer,6:184-192, 2006).

Canonical signals in the Wnt pathway are involved in stem cellproliferation. (Kim, L. & Kimmel, A. R. Current Drug Targets7:1411-1419, 2006). Glycogen synthase kinase 3 beta (GSK-3β) is a partof the Wnt signaling pathway, and its primary substrate is β-catenin.(Hagen, T et al., J. Biochem. 277(26):23330-23335). In the absence ofcanonical Wnt signaling, GSK-3β binds to β-catenin and phosphorylatesβ-catenin, thereby targeting β-catenin for ubiquitination and followedby proteosome-mediated degradation, which is mediated by AdenomatousPolyposis Coli (APC). (Id., Moon, R. T. et al., Science 296:1644-1646.2002). Canonical Wnt signals induce the release of β-catenin fromGSK-3β, thereby activating β-catenin. (Katoh, M & Katoh, M. Cancer BiolTher. 5(9):1059-64, 2006). β-catenin then localizes to the nucleus,where it activates gene transcription. (Id.).

In view of the foregoing, it would be advantageous to elucidate theinteraction between Wnt and PTEN signaling pathways and to provide newinsights into molecular regulation of stem cell proliferation anddifferentiation. It would also be advantageous to use such insights toprovide new methods, kits, and compositions for expanding stem cells invivo and ex vivo, which stem cells would be of the kind and quantitysufficient to transplant into a suitable recipient.

SUMMARY OF THE INVENTION

Thus, one embodiment of the invention is an ex vivo method for expandingthe number of hematopoietic stem cells (HSC) in a population ofmononuclear cells (MNC). This method comprises culturing the populationof MNCs comprising at least one HSC in an HSC expansion media for aperiod of time sufficient to expand the number of HSCs in the MNCpopulation, wherein the expanded HSCs are functional with long term,multi-lineage, repopulating potential.

An additional embodiment of the invention is a kit for expanding, exvivo, the number of hematopoietic stem cells (HSC) in a population ofmononuclear cells (MNC). The kit comprises a GSK-3β inhibitor, andinstructions for the use of the inhibitor, wherein, when used, the kitprovides expanded HSCs that are functional with long term,multi-lineage, repopulating potential.

A further embodiment of the invention is a media for carrying out exvivo expansion of a stem cell in a population of MNCs. This mediacomprises a fluid media suitable for maintaining viable stem cells and aGSK-3β inhibitor present in the media at a concentration sufficient toenable expansion of the stem cell population while maintaining a longterm, multi-lineage, repopulating potential in the stem cells, whereinthe stem cells, when transplanted into a recipient, exhibit greater than5% donor repopulation.

Yet another embodiment of the invention is an ex vivo method forexpanding the number of cells capable of supporting multi-lineagerepopulation in a population of mononuclear cells (MNC). This methodcomprises culturing the population of MNCs comprising at least onehematopoietic stem cell (HSC) and at least one hematopoietic progenitorcell in an HSC expansion media for a period of time sufficient to expandthe number of cells capable of supporting multi-lineage repopulation inthe MNC population.

Another embodiment of the invention is a method for expanding apopulation of stem cells obtained from a tissue selected from the groupconsisting of peripheral blood, cord blood, and bone marrow. This methodcomprises modulating a PTEN pathway and a Wnt pathway in the populationof stem cells to expand the number of stem cells.

Another embodiment of the invention is a method for ex vivo expansion ofa substantially undifferentiated stem cell population. This methodcomprises modulating a PTEN pathway and a Wnt pathway in theundifferentiated stem cell population to expand the number ofundifferentiated stem cells without significant differentiation of thestem cell population.

Yet another embodiment of the invention is a method for ex vivoexpansion of an hematopoietic stem cell (HSC) population obtained from atissue selected from the group consisting of peripheral blood, cordblood, and bone marrow. This method comprises modulating a PTEN pathwayand a Wnt pathway in the HSC population to expand the HSC population toa sufficient quantity while maintaining a multilineage differentiationpotential in the HSC population, which is sufficient for subsequenttransplantation into a patient in need thereof.

Another embodiment of the invention is an expanded, substantiallyundifferentiated stem cell population made by a method of the presentinvention. In a related embodiment, the invention is an expanded HSCpopulation made by a method of the present invention.

An additional embodiment is a method for ex vivo expansion ofhematopoietic stem cells (HSCs) by at least 40-fold, the expanded HSCsbeing competent to reconstitute an HSC lineage upon transplantation intoa mammalian patient in need thereof. This method comprises culturing apopulation of HSCs in a suitable culture medium comprising a PTENinhibitor and a GSK-3β inhibitor.

A further embodiment of the invention is a kit for expanding anhematopoietic stem cell (HSC) population for subsequent transplantationinto a patient in need thereof. The kit comprises a PTEN inhibitor, aGSK-3β inhibitor, and instructions for the use of the inhibitors.

An additional embodiment is a media for carrying out ex vivo expansionof a stem cell population. The media comprises a fluid media suitablefor maintaining viable stem cells and PTEN and GSK-3β inhibitors presentin the media at concentrations sufficient to enable expansion of thestem cell population while maintaining a multilineage differentiationpotential in the stem cells.

A further embodiment is a method for administering an hematopoietic stemcell (HSC) to a patient in need thereof. This method comprises (a)culturing, in a suitable culture media, a sample containing an HSCpopulation in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numbersufficient to transplant into the patient; (b) removing from the culturethe PTEN and Wnt pathway modulators; and (c) administering the HSCs tothe patient.

A further embodiment of the invention is a method for reconstitutingbone marrow in a patient in need thereof. This method comprises: (a)culturing, in a suitable culture media, a sample containing an HSCpopulation in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numbersufficient to transplant into the patient; (b) removing from the culturethe PTEN and Wnt pathway modulators; and (c) administering the HSCs tothe patient.

Another embodiment is a method for expanding a population ofhematopoietic stem cells (HSCs). This method comprises culturing apopulation of HSCs under conditions sufficient to result in an expansionof the HSC population by at least 40-fold, wherein the expandedpopulation of HSCs is suitable for transplantation into a mammal in needthereof.

Yet another embodiment is a method for treating a patient in need of atransplant selected from the group consisting of a bone marrowtransplant, a peripheral blood transplant, and an umbilical cord bloodtransplant. This method comprises administering to the patient apopulation of HSCs obtained by a method of the present invention.

A further embodiment is a method for expanding a population ofhematopoietic stem cells (HSCs) comprising: (a) obtaining from a mammala tissue sample comprising an HSC population; (b) expanding, in vitro,the HSC population from the sample, wherein: (i) the HSC populationexpands by at least 40-fold; and (ii) the expanded HSC population hasthe ability to reconstitute an hematopoietic lineage for at least4-weeks after transplantation into a recipient.

An additional embodiment is a method for reconstituting an hematopoieticstem cell lineage in a recipient in need thereof. This method comprises:(a) obtaining from a mammal a tissue sample comprising an HSCpopulation; (b) expanding, in vitro, the HSC population from the sample,wherein: (i) the HSC population expands by at least 40-fold; and (ii)the expanded HSC population has the ability to reconstitute anhematopoietic lineage for at least 4-weeks after transplantation into arecipient in need thereof; and (c) transplanting the expanded HSCpopulation into a recipient in need thereof.

A further embodiment of the invention is a method for expanding ahematopoietic stem cell population in a mammal in need of suchexpansion. This method comprises administering to the mammal atherapeutically effective amount of a modulator of Wnt and Akt for aperiod of time sufficient to expand the HSC population by at least40-fold with HSCs that possess the ability to reconstitute anhematopoietic lineage in the mammal.

These and other aspects of the invention are further disclosed in thedetailed description and examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a series of bar graphs and fluorescence activated cell sorting(“FACS”) analyses that collectively show that loss of PTEN withconstitutively active β-catenin leads to hematopoietic stem cell (HSC)expansion with loss of early hematopoietic progenitors.

FIG. 1A is two bar graphs showing the absolute numbers (per femur+tibia)of lineage negative, Sca-1⁺Kit⁺ (LSK) cells in Scl-Cre negative controland Scl-Cre⁺ PTEN with constitutively activated β-catenin (Pten:Ctnnb1)double mutant and each single mutant bone marrow (top) and spleen(bottom) as determined by FACS analysis. (Harada, N., et al., Embo J,18(21): 5931-42 1999. Yilmaz, O. H., et al., Nature, 441:475-82 2006.Zhang, J., et al., Nature, 441(7092): 518-22 2006.) Mice are at 10 dayspost-induction of Tamoxifen. Reduction of LSK cells in double mutantbone marrow with expansion in the spleen is indicative of mobilizationfrom bone marrow to spleen. Scl-Cre is an HSC-specific Tamoxifeninducible Cre-recombinase used to achieve conditional knockout of LoxPflanked (floxed) Pten and Ctnnb1 alleles. (Gothert, J. R., et al.,Blood, 105(7): 2724-2732, 2005.)

FIGS. 1B-1E show representative results of FACS analysis of lineagenegative, Sca-1⁺Kit⁺ (LSK) cells in Scl-Cre negative control (B and D)and Scl-Cre⁺ PTEN with constitutively activated β-catenin (Pten:Ctnnb1)double mutant (C and E) bone marrow and spleen as indicated. Boxes onthe left show Sca-1⁻Kit⁺ (early hematopoietic progenitor cells), andboxes on the right show Sca-1⁺Kit⁺ (LSK) cells. Cells were pre-gated onlive, lineage negative cells. Cells were collected from mice at 6 weekspost-induction of Tamoxifen.

FIGS. 1F and G are bar graphs showing the absolute number of LSK cellsper femur and tibia in control, Ctnnb1, Pten, and Pten:Ctnnb1 doublemutant bone marrow (F) and spleen (G) at 6 weeks post-induction. Whilethe percentage of LSKs is increased in double mutants (see FIG. 1C), lowcellularity of bone marrow from double mutants yields only moderatelyincreased absolute numbers compared to control.

FIGS. 1H and I are bar graphs and FACS analysis, respectively, ofpercentage of LSK cells, which are Flk2⁻ (indicating long-termreconstituting (LT)-HSCs) in control, Ctnnb1, Pten, and Pten:Ctnnb1mutant bone marrow at 6 weeks post-induction. Ctnnb1 single mutants arenot significantly different from controls at this time point (data notshown). Boxes in FIG. 1I indicate Flk2⁻ (LT HSC) cells.

FIG. 1J is a set of FACS analyses of CD45 in leukemic Pten:Ctnnb1 mutantbone marrow. CD45 (high) blast crisis cells are indicated (blue box,left panel). No blast cell population is observed in control or Ctnnb1mutants while a minor population has been observed in 1 of 1 Pten singlemutant mice at 6 weeks post-induction (data not shown). The right panelshows LSK analysis of leukemic Pten:Ctnnb1 mutant mouse bone marrow.Note the conversion to blast cells (lower left) with only a remnant LSKpopulation (compare to FIG. 1C).

FIG. 1K is a bar graph showing early hematopoietic progenitors definedby FACS analysis in control, Ctnnb1, Pten, and Pten:Ctnnb1 double mutantbone marrow. Common myeloid progenitor (CMP); granulocyte-monocyteprogenitor (GMP); megakaryocyte-erythrocyte progenitor (MEP); and commonlymphoid progenitor (CLP).

FIGS. 2A-J are a series of photographs, bar graphs, and FACS analysesthat collectively show that double mutant HSCs expand dramatically invitro and in vivo but fail to differentiate.

FIG. 2A is a series of photographs showing 100 LSK cells isolated fromcontrol, active β-catenin (Ctnnb1), Pten mutant, and double mutant(Pten:Ctnnb1) mice after 10 days in culture (original magnification100×). Cell numbers are not dramatically increased from 100 seeded LSKsin control while Ctnnb1 single mutant LSKs do not survive. In contrast,Pten single mutant LSKs exhibit greater proliferation but appear moreheterogeneous indicating more significant differentiation. The greatestand most homogeneous expansion occurs from Pten:Ctnnb1 double mutantLSKs.

FIG. 2B is a set of photographs showing LSK cells from Pten andPten:Ctnnb1 mutants at 34 days culture (original magnification 200×).(Note: wild-type control cultures do not expand beyond 4 weeks; Ctnnb1mutant cultures do not survive beyond 10 days.) Pten mutant HSC culturesappear more heterogeneous with significant cell clumping and moreirregular cell morphology. Also note the spindle-shaped adherent cells(arrows) showing differentiation. In contrast, double mutant HSCcultures exhibit consistent morphology. Therefore, while Pten singlemutant LSKs survive and expand, they have undergone more significantdifferentiation than the much more homogeneous Pten:Ctnnb1 double mutantLSKs.

FIGS. 2C and D are bar graphs showing the results of an expansionexperiment. Pten and Pten:Ctnnb1 LSK seven week cultures were countedand analyzed by FACS for maintenance of the LSK phenotype (wild-typecontrol and Ctnnb1 cultures did not survive this long in vitro). Doublemutant LSKs undergo >1,200 fold expansion vs. 50 fold for Pten singlemutant LSKs. LSK purity of cultures is significantly greater inPten:Ctnnb1 cultures maintaining the LSK phenotype in about 85% of totallive cells vs. about 50% for Pten single mutant cultures.

FIG. 2E is a FACS analysis of a 7 week culture of Pten:Ctnnb1 LSK cells(pre-gated on live, lineage negative cells). The boxed area indicatesKit⁺Sca-1⁺ (LSK) cells.

FIGS. 2F and G are FACS analyses showing a transplant engraftmentexperiment. At 5 weeks culture (see FIG. 2B), Pten and Pten:Ctnnb1 LSKcultures were re-sorted and 1000 LSK cells (CD45.2⁺) from each weretransplanted into lethally irradiated (10Gy) CD45.1⁺ recipient micealong with 2×10⁵ congenic whole bone marrow competitor cells. Becausewild-type cells did not survive 5 weeks culture, 1000 fresh wild-typeLSK cells were also transplanted as a separate control group. At 4 weekspost-transplant, there was no detectable engraftment from peripheralblood analysis of mice transplanted with either Pten or Pten:Ctnnb1 LSKcultures (data not shown). At 5 weeks post-transplant, bone marrow fromrecipient mice was analyzed for donor engraftment (CD45.2⁺ cells) anddonor LSK cells (CD45.2⁺ LSKs). FIGS. 2F and G display representativedonor engraftment (left, boxed areas indicate CD45.2⁺ donor cells) anddonor LSK cell engraftment (right, boxed areas indicate LSK cells) frombone marrow of mice transplanted with 1000 Fresh LSK cells (F) or 1000cultured Pten:Ctnnb1 LSK cells (G).

FIGS. 2H-J are bar graphs showing the quantitative analysis of donor(CD45.2⁺) cells (H), donor LSK cells (I), and fold increase in donorLSKs (J) isolated from bone marrow of recipient mice described in FIGS.2F and 2G at 5 weeks post-transplant.

FIGS. 3A-K are schematics, photographs, bar graphs, and FACS analysesdemonstrating that ex vivo pharmacological manipulation of the PTEN/Aktand Wnt/β-catenin signaling pathways cooperatively drive functional HSCexpansion.

FIG. 3A is a schematic illustrating representative members of the Wntand PTEN pathways. Inhibition of GSK-3β leads to β-catenin activationwhich blocks HSC differentiation. Inhibition of PTEN leads to Aktactivation which promotes survival. Both pathways individually have beenshown to promote HSC proliferation.

FIGS. 3B and C are photographs of HSCs. One hundred LSK Flk2⁻ cells weresorted from wild-type (C57BI/6) mice and cultured in (1) media, (2)media+1 μM CHIR99021 (GSK-3β inhibitor), (3) media+200 nM DipotassiumBis-peroxo(picolinato)oxovanadate (BpV(pic), a PTEN inhibitor), and (4)media+1 μM CHIR99021+200 nM BpV(pic). An alternative PTEN inhibitor,Shikonin, was also utilized at 200 nM alone (5) or in combination with 1μM CHIR99021 (6). Pictures are at 17 days culture (B, originalmagnification 100×) and 23 days (C, original magnification 40×).Compared to control, both inhibitors applied individually yield greaterexpansion of LSK cells indicating that GSK-3β inhibition is not strictlyequivalent to constitutive activation of β-catenin shown in Ctnnb1mutant LSKs while BpV(pic) yields similar results compared to Ptenmutant LSKs (see FIG. 2). Similar to double mutant LSKs (FIG. 2), thegreatest expansion is shown with both inhibitors present (FIG. 3B/C,panel 4).

FIG. 3D is a series of photographs showing LSK Flk2⁻ cells at 28 daysculture in the indicated media conditions (original magnification 200×).Here, significant expansion relative to control is observed with bothinhibitors present individually; however, significantdifferentiation/heterogeneity of cell morphology is observed in bothsingle inhibitor cultures, including more variable cell size/morphologyand/or differentiation to adherent, spindle-shaped cells (middlepanels). In contrast, and quite surprisingly, expansion with homogeneityis achieved when both inhibitors are present (last panel).

FIG. 3E is a FACS analysis of 28 day LSK Flk2⁻ cells cultured in mediacontaining both inhibitors (200 nM BpV(pic) and 1 μM CHIR99021). Cellswere pre-gated on live, lineage negative cells. The boxed area indicatesKit⁺Sca1⁺ (LSK) cells. Greater than 90% of LSKs retain Flk2 negativity(data not shown). The LSK Flk2⁻ phenotype is maintained with high purityin cultures containing both inhibitors.

FIG. 3F is a bar graph showing fold expansion of LSK Flk2⁻ cells after28 days culture in the indicated conditions. While both inhibitors addedindividually lead to significant expansion compared to media withouteither inhibitor, the greatest expansion (˜270 fold) is observed whenboth inhibitors are added together.

FIGS. 3G and H are bar graphs showing the % repopulation and % CD45.2⁺cells from engrafted mice. Twenty-eight day cultures (FIGS. 3D-F) werere-sorted for LSK Flk2− cells and 1000 LSK Flk2⁻ cells (CD45.2⁺) fromeach media condition were transplanted into lethally irradiated (10Gy)CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bone marrowcompetitor cells. At 4 weeks post-transplant, peripheral blood wasanalyzed for donor (G) and multi-lineage (H) engraftment. In FIG. 3G,each bar represents an individual mouse; the horizontal-dashed linerepresents the average “engraftment” of mice transplanted withcompetitor cells only and thus the limit of detectability for trueengraftment. Long-term (4 month) engraftment has not been observed from28-day cultures (data not shown). 6 of 8 mice show >1% engraftment whentransplanted with LSK Flk2− cells cultured with both inhibitors presentcompared to 4/8 with only CHIR99021 present, 0/10 with only BpV(pic)present, and 2/6 with media only. One percent or greater engraftment isa standard limit for substantial engraftment. (Zhang, C. C., et al., NatMed, 12(2): 240-5, 2006. Zhang, C. C. and H. F. Lodish, Blood, 105(11):4314-20, 2005.) Thus, while use of both inhibitors together leads togreatest expansion in LSKs (FIG. 3F), transplantation of equivalentnumbers of these cultured LSK Flk2⁻ cells also yields increasedshort-term engraftment/functionality when cultured with both inhibitorscompared to no or either single inhibitor only.

FIG. 3I is a bar graph showing the fold expansion of LSK Flk2⁻ cellsafter 9 days culture in. (1) media, (2) media+200 nM BpV(pic), (3)media+100 nM CHIR99021, and (4) media+200 nM BpV(pic)+100 nM CHIR99021.Because long-term engraftment was not observed from 28 day cultures(FIG. 3D-H and data not shown), LSK Flk2⁻ cells were cultured for only 9days to test if both expansion and long-term repopulation can beachieved. Similar trends are observed here to the 28 day cultures(compare to FIG. 3F) although the extent of expansion is substantiallyreduced at only 9 days compared to 28 days culture.

FIG. 3J is a FACS analysis of 9 day LSK Flk2⁻ cells cultured inmedia+200 nM BpV(pic)+100 nM CHIR99021. The boxed area indicatesKit⁺Sca-1⁺ (LSK) cells. Cells were pre-gated on live, lineage negativecells. Greater than 90% of LSKs retain Flk2 negativity (data not shown).Here, the levels of Sca-1 and Kit appear normal compared to theSca-1^((high))K^((high)) population shown from 28 day cultures (FIG.3E).

FIG. 3K is a bar graph showing % repopulation of 10-day cultured cellsin mice. Ten day cultures were transplanted into lethally irradiated(10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bonemarrow competitor cells. The total, non-adherent cell product after 10days culture of 100 initial LSK Flk2⁻ cells was transplanted per mouse.At 8 weeks post-transplant, peripheral blood was analyzed for donorengraftment. As in FIG. 3H, multi-lineage reconstitution was observedfrom all mice exhibiting true engraftment (data not shown). Each barrepresents an individual mouse; the horizontal-dashed line representsthe average ‘engraftment’ of mice transplanted with competitor cellsonly and thus the limit of detectability for true engraftment. Here, 3/7mice transplanted with LSK Flk2⁻ cells cultured in the presence of bothinhibitors exhibited 1% or greater donor engraftment vs. no micereaching this threshold in the single or no inhibitor groups.

FIG. 4 shows that ex vivo expansion of unsorted bone marrow mononuclearcells enhances functional long-term hematopoietic reconstitutionrelative to sorted, ex vivo expanded HSCs.

FIG. 4A is a logarithmic plot of CD45.2 (donor) frequency of total CD45⁺cells in peripheral blood of transplant recipients. Red line denoteslimit of detectable engraftment as determined by “engraftment” found inmice transplanted with competitor cells only.

FIG. 4B is a linear plot of CD45.2 (donor) frequency of total CD45⁺cells in peripheral blood of transplant recipients. Putative HSCs wereidentified by fluorescence activated cell sorting (FACS) based uponcell-surface markers, including lineage marker negative, Sca-1⁺, c-Kit⁺,Flk2⁻ (LSKF⁻), sorted and cultured for 14 days. Bone marrow mononuclearcells (MNCs) were also fractionated and the concentration of LSKF⁻ cellswas determined. MNCs containing a known quantity of LSKF⁻ cells werecultured for 14 days. After 14 days, the cellular product of thesecultures was transplanted into lethally-irradiated recipients at adosage corresponding to an original input into culture of 100 LSKF⁻cells per mouse for sorted cultures and MNCs containing 5 LSKF⁻ cellsper mouse for unsorted cultures. In addition, 100 freshly isolated,sorted LSKF⁻ cells per mouse and freshly isolated MNCs containing 5LSKF⁻ cells per mouse were transplanted into two additional groups.1×10⁵ competitor bone marrow cells congenic with the hosts (CD45.1⁺)were included per mouse. At 4 weeks post-transplant, peripheral bloodwas collected from each transplant recipient, and donor vs. host derivedhematopoietic cells were determined by FACS analysis.

FIG. 4C shows the percentage of donor derived peripheral blood cells(CD45.2⁺) contributing to the main hematopoietic lineages (B lymphoid, Tlymphoid, and myeloid cells) from transplant recipients described inFIGS. 4A and 4B at 4 weeks post-transplantation.

FIGS. 4D-4F show repopulation data obtained from peripheral bloodsamples from transplant recipients described in FIGS. 4A-4C at 16 weekspost-transplant.

FIGS. 4G-4H show the results of a secondary transplantation. At 16 weekspost-transplant, mice transplanted with MNCs containing 5 LSKF⁻ cellscultured for 14 days described in FIG. 4A-4F were sacrificed, and bonemarrow was isolated. A secondary transplantation was performed on newgroups of lethally irradiated mice by transplanting 1×10⁶ bone marrowcells from the original transplant group per mouse. At 4 weekspost-transplant, peripheral blood was collected from each transplantrecipient and donor-derived repopulation was determined as in FIGS.4A-4B.

FIG. 4I shows the percentage of donor derived peripheral blood cells(CD45.2⁺) contributing to the main hematopoietic lineages fromtransplant recipients described in FIG. 4G-4H at 4 weekspost-transplant.

FIGS. 4J-4L show repopulation data obtained from peripheral bloodsamples from transplant recipients described in FIGS. 4G-4I at 16 weekspost-transplant.

FIGS. 4M-4N show representative FACS plots of donor (CD45.2) vs. host(CD45.1) cells obtained from peripheral blood samples from recipientsdescribed in FIGS. 4J-4K.

FIG. 5 shows that culturing with the small-molecule inhibitor of GSK-3β,CHIR99021, enhances long-term engraftment of ex vivo expanded HSCs.

FIG. 5A is a logarithmic plot of CD45.2 (donor) frequency of total CD45⁺cells in peripheral blood of transplant recipients at 4 weekspost-transplant. FIG. 5B is a linear plot of the same. Sorted LSKF⁻cells and MNCs with a known quantity of LSKF⁻ cells were cultured andtransplanted as described in FIG. 4A. Cultures contained media alone ormedia with 250 nM CHIR99021 for each group.

FIG. 5C shows the percentage of donor derived peripheral blood cells(CD45.2⁺) contributing to the main hematopoietic lineages fromtransplant recipients described in FIGS. 5A-5B at 4 weekspost-transplant.

FIGS. 5D-5F the repopulation data obtained from peripheral blood samplesfrom transplant recipients described in FIGS. 5A-5C at 16 weekspost-transplant.

FIG. 6 shows that the ex vivo expansion protocol allows for eliminationof bone marrow rescue cells and yields engraftment equivalent to aone-hundred fold greater dosage of freshly isolated cells.

FIG. 6A shows CD45.2 (donor) frequency of total CD45⁺ cells inperipheral blood of transplant recipients at 4 weeks post-transplant.Mice transplanted with freshly isolated MNCs containing 5 LSK⁻ cells(indicated by “X”) do not survive beyond 2-3 weeks post-transplantpreventing measurement of engraftment. For this experiment, MNCs with aknown quantity of putative HSCs were cultured with and without CHIR99021for 14 days. After 14 days, the cellular product of these cultures wastransplanted into lethally-irradiated recipients at a dosagecorresponding to an original input into culture of MNCs containing 5LSKF⁻ cells. per mouse. Freshly isolated MNCs containing either 5 or 500LSKF− cells were also transplanted into 2 additional lethally irradiatedgroups of mice. No rescue/competitor bone marrow cells were included.

FIG. 6B shows the percentage of donor derived peripheral blood cells(CD45.2⁺) contributing to the main hematopoietic lineages fromtransplant recipients described in FIG. 6A at 4 weeks post-transplant.

FIGS. 6C-6D show repopulation data obtained from peripheral bloodsamples from transplant recipients described in FIGS. 6A-6B at 16 weekspost-transplant.

FIG. 6E shows the results of a secondary transplant. At 16 weekspost-transplant, mice transplanted with MNCs containing 5 or 500 LSKF⁻cells freshly isolated or cultured for 14 days described in FIGS. 6A-6Dwere sacrificed and bone marrow isolated. A secondary transplantationwas performed on new groups of lethally irradiated mice by transplanting1×10⁶ bone marrow cells from the original transplant group per mouse. At4 weeks post-transplant, peripheral blood was collected from eachtransplant recipient and donor-derived repopulation was determined. Micetransplanted with freshly isolated MNCs containing 5 LSKF⁻ cells(indicated by “X”) do not survive beyond 2-3 weeks post-transplant, thuspreventing secondary transplantation.

FIG. 6F shows the percentage of donor derived peripheral blood cells(CD45.2⁺) contributing to the main hematopoietic lineages fromtransplant recipients described in FIG. 6E at 4 weeks post-transplant.

FIGS. 6G-6H show repopulation data obtained from peripheral bloodsamples from transplant recipients described in FIGS. 6E-6F at 16 weekspost-transplant.

FIG. 7 shows ex vivo expansion of human HSCs. In FIG. 7A, bone marrowand mobilized peripheral blood was collected from human patients.Putative HSCs (CD34⁺ CD38⁻ cells) were identified by FACS analysis. Exvivo expansion was performed with and without CHIR99021. After 14 daysculture, the cellular product of these cultures was analyzed todetermine the expansion of CD34⁺ CD38⁻ cells. FIGS. 7B-7C arerepresentative FACS plots of CD34⁺ CD38⁻ cells prior to (FIG. 7B) andfollowing (FIG. 7C) ex vivo expansion.

FIG. 8 shows a β-cat-pS552 immunoassaying of homed GFP-HSCs. Detectionof β-cat-pS552⁺ (red) cells adjacent or close to N-cadherin-LacZ⁺ (blue)osteoblasts which have been identified with the HSC niche (Xie, Y. etal. Detection of functional haematopoietic stem cell niche usingreal-time imaging. Nature 457, 97-101 (2009); Zhang, J. et al.Identification of the haematopoietic stem cell niche and control of theniche size. Nature 425, 836-841 (2003)). “BM” indicates bone marrow.

FIG. 9 shows the percent of Mac-1+ Gr1+ myeloid cells in bone marrow andspleen at 8-9 weeks post-induction (wpi) in control, single and doublemutants as determined by FACS. Results are graphed as mean±SD.

FIG. 10 shows that double mutant mice lose early myeloid progenitors asmutant HSCs predominate. Data shown relate to lethally irradiatedrecipient mice previously transplanted with 1,000 LSK Flk2⁻ cellsderived from control, single and double mutant donors +200,000 congenicrescue bone marrow cells. FIG. 10A shows FACS diagrams of LSK cells(right blue boxes) and myeloid progenitors (left blue boxes) in control,single and double mutant bone marrow (top panels) and spleen (bottompanels) as indicated. As used herein, β-cat^(Act) is usedinterchangeably with Ctnnb1, and Pten:β-cat^(Act) is usedinterchangeably with Pten:Ctnnb1. Mice were at 9 or 10 wpi as indicated.Note the LS^(Los)K^(Mid) population in double mutants at 9 wpi (redarrows). FIG. 10B shows FACS analysis of early hematopoietic progenitorsin control, single and double mutant bone marrow at 9 wpi. FIGS. 10C and10D shows the absolute number of bone marrow (per tibia and femur) (FIG.10C) or spleen (FIG. 10D) LSK cells and early hematopoietic progenitorsin control, single, and double mutants at 9-10 wpi. Note the collapse ofLSK and early progenitor populations in double mutant bone marrow (redarrows) with conversion to a dominant “blast” population (see also FIG.12). FIG. 10E shows percent donor engraftment at 9 wpi oflethally-irradiated recipient mice previously transplanted with 1,000LSK Flk2⁻ cells derived from control, single and double mutant donors+200,000 congenic rescue bone marrow cells. FIG. 10F shows theEGFP-reporter expression of LSK Flk2⁻ cells in control, single anddouble mutants with the Z/EG transgenic reporter construct at 9 wpi.

FIG. 11 shows Ctnnb1 (β-cat^(Act)) HSCs undergo apoptosis whereasβ-catenin deletion prevents PTEN-deficiency-induced HSC expansion butnot myeloproliferative disorder (MPD). To obtain the results shown inFIG. 11A, 1,000 LSK Flk2⁻ cells per well were sorted from bone marrowisolated from uninduced control, Pten, Ctnnb1 (β-cat^(Act)) andPten:Ctnnb1 (Pten:β-cat^(Act)) mice. Within 12 hours of sorting, OHT wasadded to the cultures for a final concentration of 1 μM. Culturesdepicted at 4 days post-in vitro induction. FIG. 11B shows control andCtnnb1 (β-cat^(Act)) cultures as described in FIG. 11A at 48 hourspost-in vitro induction. FIG. 11C shows representative FACS plotsdistinguishing live (Sytox Green negative) from dead (Sytox Greenpositive) cells. Cultures from FIG. 11B were stained with Sytox Greenand Annexin V according to manufacturer's instructions (VybrantApoptosis Kit #9, Invitrogen) and analyzed by FACS. Live cells werefurther gated for Annexin V positive (apoptotic) cells. Numbers withingates represent the average±standard deviation from 3 independentexperiments. FIG. 11D shows the absolute number of LSK cells and earlyprogenitors in spleen as determined by FACS analysis. Mice weretransplanted with control, β-cat^(−/−), Pten, and Pten:β-cat^(−/−) micebone marrow as indicated; analysis is at 10 wpi. FIGS. 11E-G show thepercent of Gr1⁺ Mac-1⁺ cells (FIG. 11E), B-cells (FIG. 11F), and T-cells(FIG. 11G) in bone marrow of mice described in (FIG. 11D) as determinedby FACS (see FIG. 20).

FIG. 12 shows that Leukemia development and niche disruption in doublemutants. FIG. 12A shows a Kaplan-Meier survival curve for control,single and double mutants (as indicated in the figure legend) followingtamoxifen induction (Scl-Cre system unless otherwise specified). FIG.12B shows H&E stained sections of control and double mutant bone marrowat 9 wpi. White arrow indicates grossly normal cellularity in trabecularbone area. FIG. 12C shows FACS analysis of control, single and doublemutant bone marrow at 10 wpi demonstrating typical CD45 expression. NoteCD45^(High) blast cells (blue box) only mainly appear in double mutants.Blast cells from double mutants were further analyzed for cell surfacemarker expression of the T-cell specific marker, CD3.

FIG. 13 shows that Ex vivo expansion of HSCs is enhanced by inhibitionof GSK3β. For the experimental results shown in FIG. 13A, sorted LSKFlk2⁻ cells and unsorted MNCs containing a known quantity of LSK Flk2⁻cells (CD45.2⁺) were cultured for 14 days in ST media with and withoutCHIR99021. The cultured product of 100 sorted or 5 unsorted LSK Flk2⁻cells per mouse were transplanted into lethally irradiated recipients(CD45.1⁺). 5 freshly isolated, unsorted LSK Flk2⁻ cells per mouse weretransplanted into a separate group. 1×10⁵ freshly isolated CD45.1⁺competitor/radioprotective cells were also added per mouse. Peripheralblood analysis of recipients at 16 weeks post-transplant depicts %chimerism. FIG. 13B shows the percentage of donor-derived peripheralblood cells (CD45.2⁺) contributing to the main hematopoietic lineages (Blymphoid, T lymphoid, and myeloid cells) from transplant recipientsdescribed in (FIG. 13A) at 16 weeks post-transplantation. FIG. 13C showsrepresentative FACS plots of donor (CD45.2) vs. host (CD45.1) cellsobtained from peripheral blood samples at 16 weeks post-transplant fromrecipients described in FIG. 13A.

FIG. 14 shows abundant β-cat-pS552⁺ cells in double mutant spleen.Spleen sections stained with β-cat-pS552 antibody in control, single anddouble mutants at 3 dpi using Mx1-Cre system. Original magnification400× (upper panels) and 1000× (lower panels).

FIG. 15 shows trichofolliculoma in double mutants using Mx1-Cre mediatedconditional knockout. Abdomen of Mx1-Cre+ Pten:Ctnnb1 (Pten:β-cat^(Act))mutant (left panel, control mouse at left). H&E stained section of hairfollicle tumor showing multiple, well-developed but densely packed hairfollicles in cross section (right panel).

FIG. 16 shows vascular niche disruption by splenic fibrosis in doublemutants. FIG. 16A shows whole spleen isolated from control, single anddouble mutants at 9 wpi. Three examples of double mutant spleenexhibiting mild to severe fibrosis are shown. Scale bar indicates 1 cm.FIG. 16B shows Masson's Trichrome stained sections of control, singleand double mutant spleens at 9 wpi. Red arrows indicate examples ofcollagen fibers (light blue).

FIG. 17 shows the number of different types of LSK cells and earlyprogenitors, as determined by FACS (see FIG. 10) except that primarymutant mice were utilized here instead of transplant recipients as inFIG. 10. Absolute number of bone marrow (per tibia and femur) (FIG. 17A)or spleen (FIG. 17B) LSK cells and early hematopoietic progenitors incontrol, single and double mutants at 9-10 wpi. Note the collapse of LSKand early progenitor populations in double mutant bone marrow (redarrows) with conversion to a dominant “blast” population. Compare toFIG. 10.

FIG. 18 shows that Ctnnb1 (β-cat^(Act)) mutant HSCs are not maintainedin vivo. LSK Flk2⁻ cells were sorted from Scl-Cre negative control andCtnnb1 (β-cat^(Act)) mutants at 2 and 16 wpi and genotyped for deletionof exon 3. Primers utilized were: 5′-CGTGGACAATGGCTACTCAA-3′ (forward)(SEQ ID NO: 1) and 5′-TGTCAGCTCAGGAATTGCAC-3′ (reverse) (SEQ ID NO: 2)to yield wild-type (911 bp) and ΔExon 3 alleles (683 bp). Note that micewith the dominant β-cat^(Act) allele are all heterozygous for thisallele.

FIG. 19 shows the functional reversibility of myeloid differentiationblockage in double mutant HSCs. To obtain the experimental results shownin FIG. 19A, LSK Flk2⁻ cells were sorted from uninduced control, Pten,and Pten:Ctnnb1 (Pten:β-cat^(Act)) mice into an HSC expansion mediacontaining 0.25 μM 4-hydroxy-tamoxifen (OHT) and cultured for 3 days.Cultures were transduced with lentiviral vector control and vectorexpressing shRNA targeting β-catenin transcripts. Colony forming unit(CFU) assays were performed on day 6. Images depict typical coloniesfrom control, Pten, and Pten:Ctnnb1 (Pten:β-cat^(Act)) culturestransduced with control vector (left panels) and vector expressing shRNAtargeting β-catenin (right panels). Scale bar indicates 0.5 mm. FIG. 19Bshows the quantification of colonies by type from FIG. 19A includingearly erythoid progenitors (BFU-E, burst-forming unit-erythroid),granulocyte-monocyte progenitors (CFU-GM, colony formingunit-granulocyte/monocyte), and mixed early myeloid progenitors(CFU-GEMM, granulocyte/erythroid/macrophage/megakaryocyte). Large CFU(>0.5 mm diameter), which are further characterized in FIG. 19C and formonly from double mutant cultures transduced with control vector, aredesignated as primitive CFU. FIG. 19C shows panels depictingrepresentative plots of CD3 expression in control and Pten:Ctnnb1(Pten:β-cat^(Act)) cells transduced with control vector andPten:β-cat^(Act) cells transduced with vector expressing shRNA targetingβ-catenin transcripts. CFU were harvested, disaggregated intosingle-cell suspension and subjected to FACS analysis for CD3expression. Average percentage of CD3⁺ cells from 3 experiments±S.D. areshown.

FIG. 20 shows hematopoietic lineage defects and leukemogenesis in singlevs. double mutants. As in FIG. 10, mice here refer to transplantrecipients of 1,000 LSK Flk2− cells derived from control, single anddouble mutants as indicated along with 2×10⁵ congenic rescue bone marrowcells. FIG. 20A shows the percent of immature (B220^(Low), IgM⁺), mature(B220^(High), IgM⁺) and Pre-Pro B (B220^(Low), IgM⁻) cells in control,single and double mutant bone marrow at 8-9 wpi as determined by FACS.FIG. 20B shows FACS diagrams illustrating control and double mutant dataon T-cell lineage quantified in FIG. 20C. FIG. 20C shows percent ofCD3+, double and single positive T cells in control, single and doublemutant bone marrow at 8-9 wpi. Note the logarithmic scale. FIGS. 20D-Eshow Double Negative (DN) populations in control, single and doublemutant thymus at 8-9 wpi. Representative FACS plots of control (upperpanel) and double mutant (lower panel) thymus are shown in FIG. 20D.Note the logarithmic scale in FIG. 20E. FIGS. 20E-G show double andsingle positive thymocyte populations from control, single and doublemutants. Representative FACS plots of control (left panel) and doublemutant (right panel) thymus (FIG. 20F). Results are graphed as mean±SD(FIG. 20G).

FIG. 21 shows that PI3K inhibition reverses ex vivo HSC expansion andinhibits CHIR99021's ability to enhance this expansion. Bone marrow MNCswere cultured for 10 days in an HSC expansion media with and without 250nM CHIR99021, along with the indicated concentrations of PI3K inhibitor(NVP-BEZ235) (Maira, S. M. et. al. Identification and characterizationof NVP-BEZ235, a new orally available dual phosphatidylinositol3-kinase/mammalian target of rapamycin inhibitor with potent in vivoantitumor activity. Molecular cancer therapeutics 7, 1851-1863 (2008).),and then subjected to FACS analysis to determine expansion of LSK Flk2⁻cells.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is an ex vivo method for expanding thenumber of hematopoietic stem cells (HSC) in a population of mononuclearcells (MNC). This method comprises culturing the population of MNCscomprising at least one HSC in an HSC expansion media for a period oftime sufficient to expand the number of HSCs in the MNC population,wherein the expanded HSCs are functional with long term, multi-lineage,repopulating potential.

As used herein, “expand”, “expanding” and like terms means to increasethe number of stem cells in the population relative to the number ofstem cells in the original population in vitro, in vivo or ex vivo usingany of the methods disclosed herein. Preferably, the expansion is atleast 40-fold compared to the original number of stem cells in thepopulation. More preferably, the expansion is at least 80-fold,100-fold, 150-fold, 200-fold, 250-fold, or 270-fold compared to theoriginal number of stem cells.

In the present invention, “stem cells” mean cells that possess theability to give rise to many different types of cells and which have theability to self-renew. Representative, non-limiting examples of stemcells according to the present invention include bronchioalveolar stemcells (BASCs), bulge epithelial stem cells (bESCs), corneal epithelialstem cells (CESCs), cardiac stem cells (CSCs), epidermal neural creststem cells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitorcells (EPCs), hepatic oval cells (HOCs), hematopoetic stem cells (HSCs),keratinocyte stem cells (KSCs), mesenchymal stem cells (MSCs), neuronalstem cells (NSCs), pancreatic stem cells (PSCs), retinal stem cells(RSCs), and skin-derived precursors (SKPs).

Hematopoietic stem cells or HSCs, for example, have the ability toself-renew (i.e., expand) and can give rise to all the types ofprogenitor cells (such as, e.g., CMP, GMP, MEP and CLP) and ultimatelyall the types of blood cells (such as e.g., red blood cells, Blymphocytes, T lymphocytes, natural killer cells, neutrophils,basophils, eosinophils, monocytes, macrophages, and platelets) in thehematopoietic system.

As used herein, “mononuclear cells” or “MNC” mean blood cells that havea one-lobed nucleus. MNCs include without limitation monocytes,lymphocytes, plasma cells, macrophages, and mast cells.

As used herein, “HSC expansion media” means any media suitable forexpanding the number of HSC population in a culture. It includes withoutlimitation, the particular media disclosed in the Examples.

As used herein, cells with “long term, multi-lineage repopulatingpotential” means cells that are capable of repopulating many differenttypes of blood cells in irradiated recipients upon transplantationand/or cells that possess high proliferative potential in vitro.

In one aspect of this embodiment, this method provides HSCs that, upontransplant into a recipient, exhibit greater than 5% donor repopulation,such as greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90% donor repopulation. Preferably, the methodprovides HSCs that, upon transplant into a recipient, exhibit greaterthan 25%, 35%, 45%, or 60% donor repopulation. Preferably, the recipientis a mammal, for example, a primate, such as a human; or laboratoryanimals such as mice, rats, dogs, and pigs. In the present invention,“recipient” is used interchangeably with “patient.”

In another aspect of this embodiment, the HSC expansion media comprisesa modulator of the Wnt pathway. Preferably, the modulator of the Wntpathway down-regulates GSK-3β. As used herein, “down-regulating” GSK-3βmeans decreasing or inhibiting the expression or the function of GSK-3β.

In the present invention, “a modulator of a Wnt Pathway” (or “Wntpathway modulator”) is any agent that regulates the activity of anymember of the Wnt pathway, which results in, e.g., increased β-cateninexpression in a stem cell, and/or increased β-catenin function in a stemcell, and/or increased β-catenin localization to a nucleus of a stemcell. A modulator of the Wnt pathway may act upstream or downstream ofWnt. Preferably, the modulator acts at GSK-3β. Representative,non-limiting examples of members of the Wnt pathway, include Wnt,seven-transmembrane Frizzled (Fz), the single-pass, LDL receptor-relatedproteins (LRP) 5/6, Axin, Dishevelled, glycogen synthase kinase 3 beta(GSK-3β), adenomatous polyposis coli (APC), and β-catenin. Inhibition ofGSK-3β leads to Akt activation which promotes survival.

In a preferred embodiment, the modulator of the Wnt pathway is areversible GSK-3β inhibitor selected from the group consisting of asmall molecule, a biologic, an antisense RNA, a small interfering RNA(siRNA), and combinations thereof. As used herein, “reversible” meansthat the effect of the down-regulation is not permanent.

Preferably, the reversible GSK-3β inhibitor is a small molecule.Examples of reversible GSK-3β inhibitors include without limitation,Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, or combinations thereof.Preferably, the GSK-3β inhibitor is CHIR99201.

In the present invention, the term “small molecule” includes anychemical or other moiety, other than polypeptides and nucleic acids,that can act to affect biological processes, particularly to modulatemembers of the Wnt and PTEN pathways. Small molecules can include anynumber of therapeutic agents presently known and used, or that can besynthesized in a library of such molecules for the purpose of screeningfor biological function(s). Small molecules are distinguished frommacromolecules by size. The small molecules of the present inventionusually have a molecular weight less than about 5,000 daltons (Da),preferably less than about 2,500 Da, more preferably less than 1,000 Da,most preferably less than about 500 Da.

Small molecules include without limitation organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such as nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds,imidizoles, and phenols. An organic compound as used herein alsoincludes nitrated organic compounds and halogenated (e.g., chlorinated)organic compounds.

Preferred small molecules are relatively easier and less expensivelymanufactured, formulated or otherwise prepared. Preferred smallmolecules are stable under a variety of storage conditions. Preferredsmall molecules may be placed in tight association with macromoleculesto form molecules that are biologically active and that have improvedpharmaceutical properties. Improved pharmaceutical properties includechanges in circulation time, distribution, metabolism, modification,excretion, secretion, elimination, and stability that are favorable tothe desired biological activity. Improved pharmaceutical propertiesinclude changes in the toxicological and efficacy characteristics of thechemical entity.

As used herein, the term “biologic” means products derived from livingsources as opposed to a chemical process. Non-limiting examples of a“biologic” include proteins, conditioned media, and partially purifiedproducts from tissues.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein. In the present invention, these terms mean alinked sequence of amino acids, which may be natural, synthetic, or amodification or combination of natural and synthetic. The term includesantibodies, antibody mimetics, domain antibodies, Iipocalins, targetedproteases, and polypeptide mimetics. The term also includes vaccinescontaining a peptide or peptide fragment intended to raise antibodiesagainst the peptide or peptide fragment.

“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA,IgD, or IgE, or fragments or derivatives thereof, including Fab,F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, and bifunctional antibodies. The antibody may be amonoclonal antibody, polyclonal antibody, affinity purified antibody, ormixtures thereof, which exhibits sufficient binding specificity to adesired epitope or a sequence derived therefrom. The antibody may alsobe a chimeric antibody. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.The antibody may be a human or humanized antibody. These and otherantibodies are disclosed in U.S. Published Patent Application No.20070065447.

Other antibody-like molecules are also within the scope of the presentinvention. Such antibody-like molecules include, e.g., receptor traps(such as entanercept), antibody mimetics (such as adnectins, fibronectinbased “addressable” therapeutic binding molecules from, e.g., CompoundTherapeutics, Inc.), domain antibodies (the smallest functional fragmentof a naturally occurring single-domain antibody (such as, e.g.,nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15;64(8):2853-7)).

Suitable antibody mimetics generally can be used as surrogates for theantibodies and antibody fragments described herein. Such antibodymimetics may be associated with advantageous properties (e.g., they maybe water soluble, resistant to proteolysis, and/or be nonimmunogenic).For example, peptides comprising a synthetic beta-loop structure thatmimics the second complementarity-determining region (CDR) of monoclonalantibodies have been proposed and generated. See, e.g., Saragovi et al.,Science. Aug. 16, 1991; 253(5021):792-5. Peptide antibody mimetics alsohave been generated by use of peptide mapping to determine “active”antigen recognition residues, molecular modeling, and a moleculardynamics trajectory analysis, so as to design a peptide mimic containingantigen contact residues from multiple CDRs. See, e.g., Cassett et al.,Biochem Biophys Res Commun. Jul. 18, 2003; 307(1):198-205. Additionaldiscussion of related principles, methods, etc., that may be applicablein the context of this invention are provided in, e.g., Fassina,lmmunomethods. October 1994; 5(2):121-9.

Targeted proteases are polypeptides which are capable of, e.g.,substrate-targeted inhibition of post-translational modification such asdisclosed in, e.g., U.S. Patent Application Publication No. 20060275823.

In general, a polypeptide mimetic (“peptidomimetic”) is a molecule thatmimics the biological activity of a polypeptide, but that is notpeptidic in chemical nature. While, in certain embodiments, apeptidomimetic is a molecule that contains no peptide bonds (that is,amide bonds between amino acids), the term peptidomimetic may includemolecules that are not completely peptidic in character, such aspseudo-peptides, semi-peptides, and peptoids.

“Antisense” molecules as used herein include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988)and van der Krol et al., BioTechniques 6:958, (1988).

Antisense molecules can be modified or unmodified RNA, DNA, or mixedpolymer oligonucleotides. These molecules function by specificallybinding to matching sequences resulting in inhibition of peptidesynthesis (Wu-Pong, November 1994, BioPharm, 20-33) either by stericblocking or by activating an RNase H enzyme. Antisense molecules canalso alter protein synthesis by interfering with RNA processing ortransport from the nucleus into the cytoplasm (Mukhopadhyay & Roth,1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding ofsingle stranded DNA to RNA can result in nuclease-mediated degradationof the heteroduplex (Wu-Pong, supra). Backbone modified DNA chemistry,which have thus far been shown to act as substrates for RNase H arephosphorothioates, phosphorodithioates, borontrifluoridates, and2′-arabino and 2′-fluoro arabino-containing oligonucleotides.

Antisense molecules may be introduced into a cell containing the targetnucleotide sequence by formation of a conjugate with a ligand bindingmolecule, as described, e.g., in WO 91/04753. Suitable ligand bindingmolecules include, but are not limited to, cell surface receptors,growth factors, other cytokines, or other ligands that bind to cellsurface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a sense or an antisenseoligonucleotide may be introduced into a cell containing the targetnucleic acid sequence by formation of an oligonucleotide-lipid complex,as described, e.g., in WO 90/10448.

The term small interfering RNA (“siRNA”) refers to small inhibitory RNAduplexes that induce the RNA interference (RNAi) pathway. (Elbashir, S.M. et al. Nature 411:494-498 (2001); Caplen, N. J. et al. Proc. Natl.Acad. Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J Cell Sci.114:4557-4565 (2001).) These molecules can vary in length (generally18-30 base pairs) and contain varying degrees of complementarity totheir target mRNA in the antisense strand. Some, but not all, siRNA haveunpaired overhanging bases on the 5′ or 3′ end of the sense strandand/or the antisense strand. The term “siRNA” includes duplexes of twoseparate strands, as well as single strands that can form hairpinstructures comprising a duplex region. As used herein, siRNA moleculesare not limited to RNA molecules but further encompass chemicallymodified nucleotides and non-nucleotides. siRNA gene-targeting may becarried out by transient siRNA transfer into cells, achieved by suchclassic methods as lipid-mediated transfection (such as encapsulation inliposome, complexing with cationic lipids, cholesterol, and/orcondensing polymers, electroporation, or microinjection). siRNAgene-targeting may also be carried out by administration of siRNAconjugated with antibodies or siRNA complexed with a fusion proteincomprising a cell-penetrating peptide conjugated to a double-stranded(ds) RNA-binding domain (DRBD) that binds to the siRNA (see, e.g., U.S.Patent Application Publication No. 2009/0093026).

In another preferred embodiment, the method comprises culturing thepopulation of MNCs comprising at least one HSC in any of the HSCexpansion media disclosed herein, and the method provides HSCs that,upon transplant into a recipient, exhibit greater than 5%, such asgreater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90% donor repopulation, preferably, greater than60% donor repopulation.

In another aspect of this embodiment, the HSC is obtained from amammalian tissue selected from the group consisting of cord blood,peripheral blood, and bone marrow.

A further aspect of this embodiment is an expanded, substantiallyundifferentiated HSC population made by any of the methods disclosedherein. Preferably, the substantially undifferentiated HSC population ismade using an HSC expansion media comprising a modulator of the Wntpathway.

A stem cell population is “substantially undifferentiated” if asufficient number of cells in that population retain the ability toself-renew and can give rise to various differentiated cell types whentransplanted into a recipient, for example, in the case of an HSCpopulation, repopulating the HSC lineage when transplanted. As usedherein, “without significant differentiation” means the expanded stemcell population has a sufficient number of cells that maintain amulti-lineage differentiation potential that the full scope of a targetstem lineage may be regenerated upon transplantation of the expandedstem cell population into a recipient. Thus, e.g., in the case of an HSCpopulation, the expanded HSC population, when transplanted into arecipient, is capable of regenerating the entire hematopoietic celllineage.

An additional embodiment of the invention is a kit for expanding, exvivo, the number of hematopoietic stem cells (HSC) in a population ofmononuclear cells (MNC). The kit comprises a GSK-3β inhibitor, andinstructions for the use of the inhibitor, wherein, when used, the kitprovides expanded HSCs that are functional with long term,multi-lineage, repopulating potential.

In one aspect of this embodiment, the GSK-3β inhibitor is as disclosedherein. Preferably, the GSK-3β inhibitor is CHIR99201.

In another aspect of this embodiment, the kit provides HSCs that, upontransplant into a recipient, exhibit greater than 5%, such as greaterthan 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, or 90% donor repopulation, preferably, greater than 60%donor repopulation. The kit may be packaged in any convenient manner andinclude additional reagents and/or devices for carrying out its intendedpurpose.

A further embodiment of the invention is a media for carrying out exvivo expansion of a stem cell in a population of MNCs. This mediacomprises a fluid media suitable for maintaining viable stem cells and aGSK-3β inhibitor present in the media at a concentration sufficient toenable expansion of the stem cell population while maintaining a longterm, multi-lineage, repopulating potential in the stem cells, whereinthe stem cells, when transplanted into a recipient, exhibit greater than5% donor repopulation, such as greater than 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% donorrepopulation.

Yet another embodiment of the invention is an ex vivo method forexpanding the number of cells capable of supporting multi-lineagerepopulation in a population of mononuclear cells (MNC). This methodcomprises culturing the population of MNCs comprising at least onehematopoietic stem cell (HSC) and at least one hematopoietic progenitorcell in an HSC expansion media for a period of time sufficient to expandthe number of cells capable of supporting multi-lineage repopulation inthe MNC population.

As used herein, “cells capable of supporting multi-lineage repopulation”means those cells that are capable of repopulating many different typesof blood cells in irradiated recipients upon transplantation.Non-limiting examples of such cells include HSCs.

As used herein, an “hematopoietic progenitor cell” means a cell that haslost the capacity of self-renewal but is still able to give rise todifferent types of blood cells. Non-limiting examples of hematopoieticprogenitor cells include CMP, GMP, MEP, and CLP.

Another embodiment of the invention is a method for expanding apopulation of stem cells obtained from a tissue selected from the groupconsisting of peripheral blood, cord blood, and bone marrow. This methodcomprises modulating a PTEN pathway and a Wnt pathway in the populationof stem cells to expand the number of stem cells.

In the present invention, “modulating”, “modulation” and like terms meanaltering the signal transduction pathway, e.g., a protein in the PTENand/or Wnt pathways, including but not limited to lowering or increasingthe expression level of a protein, altering the sequence of such aprotein (by mutation, pre-translational or post-translationalmodification or otherwise), or inhibiting or activating such a protein(whether by binding, phosphorylation, glycosylation, translocation orotherwise). Such modulation may be achieved genetically orpharmacologically.

As used herein, “a modulator of a PTEN pathway” (or “PTEN pathwaymodulator”) is any agent that regulates the activity of any member ofthe PTEN pathway, which results in, e.g., increased β-catenin expressionin a stem cell, and/or increased β-catenin function in a stem cell,and/or increased β-catenin localization to a nucleus of a stem celland/or provides a survival signal complementary to β-catenin. Thus, amodulator of the PTEN pathway may act upstream or downstream of PTEN;preferably the modulator acts at or downstream from PTEN. Inhibition ofPTEN leads to Akt activation which promotes survival (FIG. 3A).Representative, non-limiting examples of members of the PTEN pathway,include PTEN, phosphatidylinositol 3-kinase (PI3K), the serine/threonineprotein kinase Akt, and β-catenin.

Representative non-limiting examples of PI3K modulators, particularlyPI3K activators, include pervanadate (Maude Tessier and James R.Woodgett, J. Biol. Chem., 281(33):23978-23989 (2006)), insulin (Hui, L.,et al., Brain Research, 1052(1):1-9 (2005)), insulin-like growth factor(Kenney, A. M., et al., Development, 131:217-228 (2004) and Datta, S.R., et al., Cell, 91:231-241 (1997)), platelet derived growth factor(Datta, S. R., et al., Cell 91:231-241 (1997)), carbachol (Cui, Q L, etal., Neurochem Int, 48:383-393 (2006)), nicotine (West, K. et al., J.Clinical Investigation, 111:81-90 (2003)),4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Id.),adrenomedullin (AM) (Nikitenko, L L et al., British J. Cancer, 94:1-7(2006)), lysophosphatidic acid, platelet activating factor, macrophagesimulating factor, and sphingosine-1-phosphate.

Representative non-limiting examples of Akt modulators, particularly Aktactivators, include Ro-31-8220 (Wen, H. et al., Cellular signaling,15:37-45 (2003)); Nicotine (West, K. et al., J. Clinical Investigation,111:81-90 (2003)); carbachol (Cui Q L, Fogle E & Almazan G NeurochemInt, 48:383-393 (2006)); 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) (West, K. et al., J. Clinical Investigation, 111:81-90 (2003));adrenomedullin (AM) (Nikitenko, L L et al, British J. Cancer, 94:1-7(2006)); lysophosphatidic acid; platelet activating factor, macrophagesimulating factor; sphingosine-1-phosphate; cAMP-elevating agents, suchas forskolin, chlorophenylthio-cAMP, prostaglandin-E1, and 8-bromo-cAMP(Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005)); and growthfactors, including insulin and insulin growth factor-1 (Datta, S. R., etal., Cell, 91:231-241 (1997)), and platelet derived growth factor.

Additional preferred modulators of the present invention include thosethat target mTOR, RHEB, FoxO, p27, BAD, caspase-9, or p53.Representative non-limiting examples of such modulators include mTORmodulators, particularly mTOR activators, such as phosphatidic acid (PA)(see, e.g., WO/2006/027545; Foster, D. A., Cancer Res, 67(1):1-4 (2007);and Tee et al., J. Biol. Chem. 278:37288-96 (2003)); RHEB modulators,particularly RHEB-GTPase inhibitors, such as RHEB antibodies (see, e.g.,WO/2004/048536); FoxO modulators, particularly FoxO inhibitors, such asFKH(DBD), which is a truncated version of FKHRL1 (see, e.g., Gilley, J.,et al., J. Cell Biol. 162(4):613-622 (2003)); p27 modulators,particularly p27 inhibitors, such as p27 antisense inhibitors andtriplex forming oligonucleotides, protein and peptide antagonists (see,e.g., U.S. Pat. No. 5,958,769); BAD modulators, particularly BADinhibitors, such as 14-3-3 protein (see, e.g., S. Hsu et al., MolecularEndocrinology 11 (12):1858-1867 (1997)); caspase-9 modulators,particularly caspase-9 inhibitors, such as LB-84451 (LG Life Sciences)and Z-LEHD-FMK Caspase Inhibitor (Thornberry, N. A., and Lazebnik, Y.,Science 281:1312-1316 (1998)); and p53 modulators, particularly p53inhibitors, such as Pifithrin-a and its derivatives (see, e.g., Science,Komarov et al., 285 (5434): 1733-1737 (1999), Pietrancosta et al., DrugDev Res 65:43-49 (2005)).

In one aspect of the present invention, modulating the PTEN pathwaycomprises introducing a mutation into a population of stem cells, whichmutation results in modulation of a molecule in the PTEN pathway. In thepresent invention, modulation of the PTEN pathway also includescontacting the stem cells with a modulator of a molecule in the PTENpathway that leads to β-catenin activation. Representative, non-limitingexamples of such modulators include a small molecule, a biologic, anantisense RNA, a small interfering RNA (siRNA), and combinationsthereof. This aspect of the invention further includes modulating theWnt pathway, which comprises introducing a mutation into a population ofstem cells that results in modulation of a molecule in the Wnt pathway.In the present invention modulation of the Wnt pathway also includescontacting the stem cells with a modulator of a molecule in the Wntpathway. Representative, non-limiting examples of such a modulatorinclude a small molecule, a biologic, an antisense RNA, a smallinterfering RNA (siRNA), and combinations thereof.

As used herein, “introducing a mutation” means any conventional methodfor producing an alteration in the genetic makeup of the stem cellpopulation. Non-limiting examples for introducing a mutation into a stemcell population include mutagenesis via ultra-violet light irradiation,chemical mutagenesis, targeted mutagenesis such as site directedmutagenesis of a stem cell, and creation of a transgenic mouse.

In the present invention, the phrase “modulation of a molecule in thePTEN pathway” means altering the function of a member of the PTENpathway, which altered function has an effect similar to inhibiting ordecreasing the function of PTEN. Non-limiting examples of such“modulation” include constitutively activating β-catenin, constitutivelyactivating Akt, or loss-of-function or null alleles of PTEN. The phrase“modulation of a molecule in the Wnt pathway” means blocking ordecreasing the function of a member of the Wnt pathway, which has aneffect similar to blocking or decreasing GSK-3β function. Non-limitingexamples of such modulation include constitutively activating β-cateninand loss-of-function or null alleles of GSK-3β.

“Modulators of a molecule in the PTEN pathway” are molecules that cause,directly or indirectly, activation of β-catenin. Non-limiting examplesof such molecules include those that activate β-catenin, activate Akt,activate PI3K, or inhibit PTEN. “Modulators of a molecule in the Wntpathway” are molecules that directly or indirectly block or decrease thefunction of a member of the Wnt pathway. Non-limiting examples of suchmolecules include those that activate β-catenin or that inhibit GSK-3β,Axin, or APC.

In another aspect of the present invention, modulating the PTEN and Wntpathways comprises contacting the stem cell population with a smallmolecule inhibitor of the PTEN pathway and a small molecule inhibitor ofthe Wnt pathway. Preferably, modulating the PTEN and Wnt pathwayscomprises down-regulating PTEN and GSK-3β, respectively. As used herein,“down-regulating” means inhibiting or reducing the amount of orinhibiting or decreasing the activity of PTEN and GSK-3β. Suchdown-regulation may be accomplished using, e.g. antisense RNA, siRNA,antibodies, or small molecules.

Preferably, down-regulating PTEN and GSK-3βcomprises contacting the stemcell population with: (a) a reversible PTEN inhibitor selected from thegroup consisting of a small molecule, a biologic, an antisense RNA, asmall interfering RNA (siRNA), and combinations thereof and (b) areversible GSK-3β inhibitor selected from the group consisting of asmall molecule, a biologic, an antisense RNA, a small interfering RNA(siRNA), and combinations thereof. In the present invention, geneticalteration of both the PTEN and the Wnt pathways leads to an increasedability to self-renew both in vitro as well as in vivo followinglong-term culture but a failure to differentiate and thus a failure torepopulate the hematopoietic system of transplant recipients. Incontrast, use of reversible down-regulators of both pathways, such as,e.g., bpV(pic) and CHIR99201, allows for expansion of functional HSCs,but (1) once the down-regulator is withdrawn, cultured HSCs candifferentiate unlike cultured HSCs from genetic mutants, and (2) if suchcultured HSC are transplanted, recipient animals do not develop leukemiaas genetic mutants do.

Preferably, both the reversible PTEN inhibitor and the reversible GSK-3βinhibitor are small molecules. In one aspect, the reversible PTENinhibitor is any molecule, such as a small molecule, which is capable ofinhibiting PTEN or a down-stream member of the PTEN pathway, whichinhibition leads to β-catenin activation. Preferably, the PTEN inhibitoris selected from the group consisting of shikonin, a bisperoxovanadiumcompound, SF-1751 (Semafore Pharmaceuticals), pharmaceutical saltsthereof, and combinations thereof. In this aspect, the bisperoxovanadiumcompound is selected from the group consisting of bpV(phen)2, bpV(pic),pharmaceutical salts thereof, and combinations thereof.

In the present invention, the reversible GSK-3β inhibitor is anymolecule that is capable of reversibly inhibiting GSK-3β. Preferably,such an inhibitor is selected from the group consisting ofHymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, and combinations thereof.

While the PTEN and GSK-3β inhibitors may be contacted with the stem cellpopulation in any convenient manner that achieves the desired level ofstem cell expansion, it is preferred that the inhibitors areco-administered. Moreover, multiple GSK-3β and PTEN inhibitors may becontacted with the stem cells. Furthermore, the PTEN and GSK-3βinhibitors may be contacted/administered to the stem cells in concertwith other agents suitable for promoting stem cell self renewal.Preferably, the PTEN inhibitor is bpV(pic) and the GSK-3β inhibitor isCHIR99201.

In an additional aspect of the present invention, the number of stemcells is increased by a factor of at least 40-fold. Preferably, thenumber of stem cells is increased by a factor of at least 80-fold, suchas at least 100-fold, including at least 150-fold, at least 200-fold, atleast 250-fold, or at least 270-fold. Surprisingly and unexpectedly suchlevels of stem cell expansion are achieved using the methods of thepresent invention.

As noted above, the methods of the present invention may be used toexpand any population of stem cells. Representative, non-limitingexamples of stem cells are as set forth above. Preferably, the stemcells that may be expanded according to the methods of the presentinvention may selected from hematopoietic stem cells (HSCs), endothelialprogenitor cells (EPCs), mesenchymal stem cells (MSCs), cardiac stemcells (CSCs), neuronal stem cells (NSCs), and combinations thereof. Morepreferably, the stem cells are HSCs.

Another embodiment of the invention is a method for ex vivo expansion ofa substantially undifferentiated stem cell population. This methodcomprises modulating a PTEN pathway and a Wnt pathway in theundifferentiated stem cell population to expand the number ofundifferentiated stem cells without significant differentiation of thestem cell population.

A further embodiment of the invention is a method for ex vivo expansionof an hematopoietic stem cell (HSC) population obtained from a tissueselected from the group consisting of peripheral blood, cord blood, andbone marrow. This method comprises modulating a PTEN pathway and a Wntpathway in the HSC population to expand the HSC population to asufficient quantity while maintaining a multilineage differentiationpotential in the HSC population, which is sufficient for subsequenttransplantation into a patient in need thereof.

As used herein, “obtained” from a tissue means any conventional methodof harvesting or partitioning tissue from a donor. For example, thetissue may obtained from a blood sample, such as a peripheral or cordblood sample, or harvested from bone marrow. Methods for obtaining suchsamples are well known to the artisan. In the present invention, thesamples may be fresh, i.e., obtained from the donor without freezing.Moreover, the samples may be further manipulated to remove extraneous orunwanted components prior to expansion. The samples may also be obtainedfrom a preserved stock. For example, in the case of peripheral or cordblood, the samples may be withdrawn from a cryogenically or otherwisepreserved bank of such blood. Such samples may be obtained from anysuitable donor. Preferably, the donor is a mammal, for example, aprimate, such as a human; or laboratory animals such as mice, rats,dogs, and pigs. Furthermore, the sample may be obtained from anautologous or allogeneic donor or source. Preferably, the sample isobtained from an autologous source.

In this method, “maintaining a multilineage differentiation potential”means that the expanded HSC population has the ability, whentransplanted into a patient in need of such a transplant, to regenerateall the types of progenitor cells e.g., CMP, GMP, MEP, and CLP, andultimately all the types of blood cells including, e.g., red bloodcells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils,basophils, eosinophils, monocytes, macrophages, and platelets in thehematopoietic system.

In the present invention, that quantity of expanded HSCs, which is“sufficient for subsequent transplantation” generally corresponds tothat number of HSCs, which would result in greater than about 1%engraftment after transplantation. This is one accepted measure of asuccessful transplant. In the present invention, any conventional methodmay be used to determine the % engraftment, including the one set forthin the Examples. Such a measure may be carried out with or withoutcompetitor cells, typically and preferably, without competitor cells.(Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang, C. C. and H.F. Lodish, Blood, 105(11): 4314-20, 2005).

In the above described ex vivo expansion methods, modulating the PTENand Wnt pathways may be achieved as previously set forth. Modulating thePTEN and Wnt pathways may include contacting the stem cell populationwith a small molecule inhibitor of the PTEN pathway and a small moleculeinhibitor of the Wnt pathway. Modulating the PTEN and Wnt pathways mayinclude down-regulating PTEN and GSK-3β, respectively. Preferably,down-regulating the PTEN and Wnt pathways comprises contacting the stemcell population with a reversible PTEN inhibitor and a reversible GSK-3βinhibitor as previously described. Preferably, both the reversible PTENinhibitor and the reversible GSK-3β inhibitor are small molecules.

The reversible PTEN inhibitor may be selected from the group consistingof shikonin, a bisperoxovanadium compound, SF-1751 (SemaforePharmaceuticals), pharmaceutical salts thereof, and combinationsthereof. Preferably, the bisperoxovanadium compound is selected from thegroup consisting of bpV(phen)2, bpV(pic), pharmaceutical salts thereof,and combinations thereof.

The reversible GSK-3β inhibitor may be selected from the groupconsisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, and combinations thereof.

In these ex vivo expansion methods, preferably, the PTEN inhibitor isbpV(pic), and the GSK-3β inhibitor is CHIR99201. In these methods, it ispreferred that the stem cell is selected from HSCs, endothelialprogenitor cells, (EPCs), mesenchymal stem cells (MSCs), cardiac stemcells (CSCs), neuronal stem cells (NSCs), and combinations thereof.Preferably the stem cell is an HSC. In these methods, the HSC isobtained from a mammalian, e.g., primate or human, tissue selected fromthe group consisting of cord blood, peripheral blood, and bone marrow.

In another aspect of the method for ex vivo expansion of anhematopoietic stem cell (HSC) population, the expansion of the number ofstem cells is by at least 40-fold, such as e.g., by at least 80-fold,including at least 100-fold, at least 150-fold, at least 200-fold, atleast 250-fold, or at least 270-fold.

Yet another embodiment of the present invention is an expanded,substantially undifferentiated stem cell population made by a method ofthe present invention, such as, e.g., the method for ex vivo expansionof a substantially undifferentiated stem cell population or the methodfor ex vivo expansion of an hematopoietic stem cell (HSC) population.

An additional embodiment of the present invention is a method for exvivo expansion of hematopoietic stem cells (HSCs) by at least 40-fold,wherein the expanded HSCs, are competent to reconstitute an HSC lineageupon transplantation into a mammalian patient in need thereof. Thismethod comprises culturing a population of HSCs in a suitable culturemedium comprising a PTEN inhibitor and a GSK-3β inhibitor.

In this aspect of the invention, “competent to reconstitute an HSClineage” means that the expanded HSCs, when transplanted into a suitablemammalian patient, result in greater than 1% engraftment in therecipient, which engrafted cells are able to differentiate into the celllineages necessary to have a normal functioning hematopoietic system. Inthis method, a “suitable culture medium”, “fluid media” and “media”which are used interchangeably herein, mean physiologically balancedsalt solutions that can maintain a stem cell population, for a requiredperiod of time, which solution may be supplemented with the PTEN andGSK-3β modulator/inhibitors of the present invention. Such base culturemedia are well known in the arts. A non-limiting example of a suitablebase culture medium for HSCs is StemSpan Media (Stem Cell Technologies;Cat. No. 09600), which is supplemented with 10 ug/ml Heparin, 0.5×Penicillin/Streptomycin, 10 ng/ml recombinant mouse (rm) Stem CellFactor, and 20 ng/ml rm-Thrombopoietin.

Typically, the culture media also includes from about 100 to about 1000nM of the PTEN inhibitor. The culture media may further include fromabout 50 nM to about 500 nM of the GSK-3β inhibitor. In the presentinvention, when a range is recited, any value within that range,including the endpoints, is contemplated. Preferably, the culture mediaincludes both the PTEN and the GSK-3β inhibitors at the concentrationsindicated. For example, the media may contain as the PTEN inhibitor,bpV(pic), and as the GSK-3β inhibitor, CHIR99201.

In one aspect of this embodiment, the HSCs are obtained from a mammaliantissue, preferably primate or human tissue, which is selected from cordblood, peripheral blood, and bone marrow. In this embodiment, the numberof HSCs is expanded by a factor of at least 80-fold, such as at least100-fold, including at least 150-fold, at least 200-fold, at least250-fold, or at least 270-fold.

Yet another embodiment of the present invention is a kit for expandingan hematopoietic stem cell (HSC) population for subsequenttransplantation into a patient in need thereof. The kit comprises a PTENinhibitor and a GSK-3β inhibitor as described above and instructions forthe use of the inhibitors. Preferably, in the kit, the PTEN inhibitor isbpV(pic) and the GSK-3β inhibitor is CHIR99201. The kit and thecomponents therein may be packaged in any suitable manner fordistribution and/or storage.

A further embodiment of the present invention is a media for carryingout ex vivo expansion of a stem cell population. The media comprises afluid media suitable for maintaining viable stem cells and PTEN andGSK-3β inhibitors present in the media at concentrations sufficient toenable expansion of the stem cell population while maintaining amultilineage differentiation potential in the stem cells.

In this embodiment, a “concentration sufficient to enable expansion”means the minimum concentration of the PTEN and GSK-3β inhibitors, whichare sufficient to achieve the desired level of stem cell renewal, e.g.,expansion sufficient for successful engraftment.

In one aspect of this embodiment, expansion of the number of stem cellsis by a factor selected from the group consisting of at least 40-fold,at least 80-fold, at least 100-fold, at least 150-fold, at least200-fold, at least 250-fold, or at least 270-fold.

A further embodiment of the present invention is a method foradministering an hematopoietic stem cell (HSC) to a patient in needthereof. The method comprises (a) culturing, in a suitable culturemedia, a sample containing an HSC population in the presence of amodulator of a molecule in the PTEN pathway and a modulator of amolecule in the Wnt pathway for a period of time sufficient to expandthe number of HSCs in the sample to a number sufficient to transplantinto the patient; (b) removing from the culture the PTEN and Wnt pathwaymodulators; and (c) administering the HSCs to the patient. In thisembodiment, the culture media, sample, and PTEN and GSK-3β modulatorsare previously described.

An additional embodiment of the present invention is a method forreconstituting bone marrow in a patient in need thereof. The methodcomprises culturing, in a suitable culture media, a sample containing anHSC population in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numberthat is sufficient to transplant into the patient. Next, the PTEN andWnt pathway modulators are removed from the culture. Then, the expandedHSCs are administered to the patient in any conventional manner.

In this method, “reconstituting bone marrow” means restoration of all ora portion of the bone marrow in a patient suffering from a disease inwhich normal bone marrow function has been compromised. Non-limitingexamples of such diseases include aplastic anemia, myelodysplasticsyndromes (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and bloodcancers, such as leukemia. Thus, as used herein, “reconstituted” meansthat the transplanted HSCs are able to successfully engraft in the hostand differentiate into all the cell lineages typically found in orderived from bone marrow.

In this method, “a period of time sufficient to expand the number ofHSCs” means the minimum amount of time to expand the HSCs in culture toa point where there is a sufficient number of HSCs for one or moretransplantations. Typically, such a period of time may be at least about10 days in culture. Under certain circumstances, it may be desirable toexpand the stem cell, e.g., HSC, population beyond what is required fora single transplantation. For example, it may be desirable to expand thestem cell, e.g., HSC, population to a number sufficient for multipletransplantations, such as e.g., from about 2 to about 100transplantations. In these circumstances, the excess cells may bepreserved for later use by any conventional method, such as e.g., bycryo-preservation.

As indicated previously, “a number sufficient to transplant” means theminimum number of stem cells, e.g., HSCs, necessary to achieve greaterthan 1% engraftment in a recipient. “Administering the HSCs to thepatient” means conventional methods for delivering HSCs to the patient,including but not limited to, delivering the HSCs surgically and/orintravenously. In this embodiment, the tissue the HSCs are obtainedfrom, and the PTEN and GSK-3β inhibitors are as previously described.

An additional embodiment of the present invention is a method forexpanding a population of hematopoietic stem cells (HSCs). This methodcomprises culturing a population of HSCs under conditions sufficient toresult in an expansion of the HSC population by at least 40-fold,wherein the expanded population of HSCs is suitable for transplantationinto a mammal in need thereof. In this embodiment the “conditionssufficient to result in an expansion of the HSC population” are thoseconditions that can result in expansion of HSCs in culture by, e.g., atleast 40-fold, such as, e.g., by at least 80-fold, at least 150-fold, atleast 200-fold, at least 250-fold, or at least 270-fold. “Suitable fortransplantation into a mammal” means that the number and quality of HSCsis sufficient to support greater than 1% engraftment in a mammalianrecipient, such as, e.g., a primate recipient, including an humanrecipient, in need thereof.

Yet another embodiment of the present invention is a method for treatinga patient in need of a bone marrow transplant, a peripheral bloodtransplant, or a cord blood transplant comprising administering to thepatient a population of HSCs obtained by a method disclosed herein,particularly the methods for expanding a population of hematopoieticstem cells (HSCs).

A further embodiment of the present invention is a method for expandinga population of hematopoietic stem cells (HSCs). The method comprises(a) obtaining from a mammal a tissue sample comprising an HSCpopulation; (b) expanding, in vitro, the HSC population from the sample,wherein (i) the HSC population expands by at least 40-fold; and (ii) theexpanded HSC population has the ability to reconstitute an hematopoieticlineage for at least 4-weeks, such as for example at least 8-weeks,after transplantation into a recipient. In this embodiment the “abilityto reconstitute an hematopoietic lineage” means that the expanded HSCpopulation when transplanted into a recipient will result in greaterthan 1% engraftment of HSC in a recipient. In one aspect of thisembodiment, the HSC population expands by at least 80-fold, such ase.g., at least 100-fold, including at least 150-fold, at least 200-fold,at least 250-fold, or at least 270-fold. In another aspect of thisembodiment, the mammal is a primate, including a human. Preferably, thehuman requires a peripheral blood transplant, a cord blood transplant,or a bone marrow transplant. In a further aspect, the tissue sample isobtained from a tissue selected from the group consisting of cord blood,peripheral blood, and bone marrow.

An additional embodiment of the present invention is a method forreconstituting an hematopoietic stem cell lineage in a recipient in needthereof. The method comprises(a) obtaining from a mammal a tissue samplecomprising an HSC population; (b) expanding, in vitro, the HSCpopulation from the sample, wherein: (i) the HSC population expands byat least 40-fold, such as for example, by at least 80-fold, including atleast 100-fold, at least 150-fold, at least 200-fold, at least 250-fold,or at least 270-fold, and (ii) the expanded HSC population has theability to reconstitute an hematopoietic lineage for at least 4-weeks,for example, at least 8-weeks, after transplantation into a recipient inneed thereof; and (c) transplanting the expanded HSC population into arecipient such as a mammal, including a primate or human, in needthereof.

In this embodiment, “reconstituting an hematopoietic stem cell lineage”,means that the expanded HSCs, when transplanted into a recipient resultin greater than 1% engraftment of hematopoietic cells, which are able todifferentiate into the normal hematopoietic lineages. In thisembodiment, the human recipient requires a peripheral blood transplant,a cord blood transplant or a bone marrow transplant. Thus, in a furtheraspect, the tissue sample is obtained from a tissue selected from thegroup consisting of cord blood, peripheral blood, and bone marrow. Thesample may be obtained from an autologous or allogeneic source.Preferably, the sample is obtained from an autologous source.

In the present invention, it is preferred that the expanded HSCpopulation comprises HSCs that have a phenotype selected from the groupconsisting of CD34⁻ orCD34⁺/CD38^(−/low)/Thy-1⁺/CD90⁺/Kit^(−/lo)/Lin⁻/CD133⁺VEGFR2⁺, which aremarkers for the most primitive and long-term undifferentiated humanHSCs; CD150⁺/CD48⁻/CD244⁻, which is a marker for human HSCs and theirprogenitors; and/or CD150⁻/CD48⁻/CD244⁺ and CD150⁻/CD48⁺/CD244⁺, whichare markers for non-self-renewing multipotent hematopoietic progenitors,and combinations thereof. (See, e.g., Mimeault, M., et al., Stem Cells:A Revolution in Therapeutics—Recent Advances in Stem Cell Biology andTheir Therapeutic Applications in Regenerative Medicine and CancerTherapies. Clin Pharmacol Ther., 82(3):252-64 (2007)).

The exact proportions of HSCs having these markers in the population isnot critical, so long as the expanded HSC population as a whole issufficient to result in at least 1% engraftment in a recipient.

In another embodiment, the invention is a method for expanding ahematopoietic stem cell population in a mammal in need of suchexpansion. This method comprises administering to the mammal atherapeutically effective amount of a modulator of Wnt and Akt for aperiod of time sufficient to expand the HSC population by at least40-fold with HSCs that possess the ability to reconstitute anhematopoietic lineage in the mammal.

In this method, the respective modulators of Wnt and Akt may be anymolecule, such as a small molecule, a biologic, an antisense RNA, asiRNA, or combinations thereof, which acts directly or indirectly toactivate β-catenin. Preferably, the Wnt modulator is selected from a Wntpolypeptide, QS11 (Zhang, Q. et al., PNAS, 104(18):7444-8 (2007)),2-amino-443,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine(Liu, J. et al., Angew Chem Int Ed Engl. 44(13):1987-90 (2005)),deoxycholic acid (R. Pai et al., Mol Biol Cell. 15(5):2156-63 (2004)),and combinations thereof. Preferably, the modulator of Akt is selectedfrom the group consisting of Ro-31-8220 (Wen, H. et al., Cellularsignaling, 15:37-45 (2003)); Nicotine (West, K. et al., J. ClinicalInvestigation, 111:81-90 (2003)); carbachol (Cui Q L, Fogle E & AlmazanG Neurochem Int, 48:383-393 (2006));4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (West, K. et al.,J. Clinical Investigation, 111:81-90 (2003)); adrenomedullin (AM)(Nikitenko, L L et al, British J. Cancer, 94:1-7 (2006));lysophosphatidic acid; platelet activating factor, macrophage simulatingfactor; sphingosine-1-phosphate; cAMP-elevating agents, such asforskolin, chlorophenylthio-cAMP, prostaglandin-E1, and 8-bromo-cAMP(Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005)); and growthfactors, including insulin and insulin growth factor-1 (Datta, S. R., etal., Cell, 91:231-241 (1997)), platelet derived growth factor, andcombinations thereof.

In this method, the Wnt and Akt modulators may be administered using anyregimen that effectively expands the HSC population by at least 40-foldwith HSCs that possess the ability to reconstitute an hematopoieticlineage in the mammal. Preferably, the Wnt and Akt modulators areco-administered.

In the present invention, a “therapeutically effective amount” is anamount sufficient to effect beneficial or desired results. In terms oftreatment of a mammal, a “therapeutically effective amount” of amodulator is an amount sufficient to treat, manage, palliate,ameliorate, or stabilize a condition, such as a bone marrow disease, inthe mammal. A therapeutically effective amount can be administered inone or more doses.

The therapeutically effective amount is generally determined by aphysician on a case-by-case basis and is within the skill of one in theart. Several factors are typically taken into account when determiningan appropriate dosage. These factors include age, sex and weight of thepatient, the condition being treated, the severity of the condition andthe form of the drug being administered.

Effective dosage forms, modes of administration, and dosage amounts maybe determined empirically, and making such determinations is within theskill of the art. It is understood by those skilled in the art that thedosage amount will vary with the route of administration, the rate ofexcretion, the duration of the treatment, the identity of any otherdrugs being administered, the age, size, and species of animal, and likefactors well known in the arts of medicine and veterinary medicine. Ingeneral, a suitable dose of a modulator according to the invention willbe that amount of the modulator, which is the lowest dose effective toproduce the desired effect. The effective dose of a modulator maybeadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

A modulator, particularly a Wnt or Akt modulator of the presentinvention, may be administered in any desired and effective manner: aspharmaceutical compositions for oral ingestion, or for parenteral orother administration in any appropriate manner such as intraperitoneal,subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal,vaginal, sublingual, intramuscular, intravenous, intraarterial,intrathecal, or intralymphatic. Further, a modulator, particularly a Wntor Akt modulator, of the present invention may be administered inconjunction with other treatments. A modulator, particularly a Wnt orAkt modulator, of the present invention maybe encapsulated or otherwiseprotected against gastric or other secretions, if desired.

While it is possible for a modulator, particularly a Wnt or Aktmodulator, of the invention to be administered alone, it is preferableto administer the modulator as a pharmaceutical formulation(composition). Such pharmaceutical formulations typically comprise oneor more modulators as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, one or more othercompounds, drugs, ingredients and/or materials. Regardless of the routeof administration selected, the modulator, particularly a Wnt or Aktmodulator, of the present invention is formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art. See, e.g., Remington's PharmaceuticalSciences (Mack Publishing Co., Easton, Pa.).

Pharmaceutically acceptable carriers are well known in the art (see,e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.) and The National Formulary (American Pharmaceutical Association,Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol,and sorbitol), starches, cellulose preparations, calcium phosphates(e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable carrier used ina pharmaceutical composition comprising a modulator of the inventionmust be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Carriers suitable for a selected dosage form and intended route ofadministration are well known in the art, and acceptable carriers for achosen dosage form and method of administration can be determined usingordinary skill in the art.

Pharmaceutical compositions comprising a modulator of the invention may,optionally, contain additional ingredients and/or materials commonlyused in pharmaceutical compositions. These ingredients and materials arewell known in the art and include (1) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (2)binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium starch glycolate, cross-linked sodiumcarboxymethyl cellulose and sodium carbonate; (5) solution retardingagents, such as paraffin; (6) absorption accelerators, such asquaternary ammonium compounds; (7) wetting agents, such as cetyl alcoholand glycerol monosterate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, and sodium lauryl sulfate; (10)suspending agents, such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth;(11) buffering agents; (12) excipients, such as lactose, milk sugars,polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins,cocoa butter, starches, tragacanth, cellulose derivatives, polyethyleneglycol, silicones, bentonites, silicic acid, talc, salicylate, zincoxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13)inert diluents, such as water or other solvents; (14) preservatives;(15) surface-active agents; (16) dispersing agents; (17) control-releaseor absorption-delaying agents, such as hydroxypropylmethyl cellulose,other polymer matrices, biodegradable polymers, liposomes, microspheres,aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19)adjuvants; (20) wetting agents; (21) emulsifying and suspending agents;(22), solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan; (23) propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane; (24) antioxidants; (25) agents which render theformulation isotonic with the blood of the intended recipient, such assugars and sodium chloride; (26) thickening agents; (27) coatingmaterials, such as lecithin; and (28) sweetening, flavoring, coloring,perfuming and preservative agents. Each such ingredient or material mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Ingredients and materials suitable for a selected dosage form andintended route of administration are well known in the art, andacceptable ingredients and materials for a chosen dosage form and methodof administration may be determined using ordinary skill in the art.

Pharmaceutical compositions suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules, asolution or a suspension in an aqueous or non-aqueous liquid, anoil-in-water or water-in-oil liquid emulsion, an elixir or syrup, apastille, a bolus, an electuary or a paste. These formulations may beprepared by methods known in the art, e.g., by means of conventionalpan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared by mixing theactive ingredient(s) with one or more pharmaceutically-acceptablecarriers and, optionally, one or more fillers, extenders, binders,humectants, disintegrating agents, solution retarding agents, absorptionaccelerators, wetting agents, absorbents, lubricants, and/or coloringagents. Solid compositions of a similar type maybe employed as fillersin soft and hard-filled gelatin capsules using a suitable excipient. Atablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using asuitable binder, lubricant, inert diluent, preservative, disintegrant,surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine. The tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein. They may be sterilized by, forexample, filtration through a bacteria-retaining filter. Thesecompositions may also optionally contain opacifying agents and may be ofa composition such that they release the active ingredient only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Pharmaceutical compositions for rectal or vaginal administration may bepresented as a suppository, which maybe prepared by mixing one or moreactive ingredient(s) with one or more suitable nonirritating carrierswhich are solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing suchpharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active compound may be mixed understerile conditions with a suitable pharmaceutically-acceptable carrier.The ointments, pastes, creams and gels may contain excipients. Powdersand sprays may contain excipients and propellants.

Pharmaceutical compositions suitable for parenteral administrationscomprise one or more modulator in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug containing amodulator of the present invention, it is desirable to slow itsabsorption from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility.

The rate of absorption of the drug then depends upon its rate ofdissolution which, in turn, may depend upon crystal size and crystallineform. Alternatively, delayed absorption of a parenterally-administereddrug may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms may be made by forming microencapsulematrices of the active ingredient in biodegradable polymers. Dependingon the ratio of the active ingredient to polymer, and the nature of theparticular polymer employed, the rate of active ingredient release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The following examples are provided to further illustrate the methodsand compositions of the present invention. These examples areillustrative only and are not intended to limit the scope of theinvention in any way.

Examples Example 1 Loss of PTEN with Constitutively Active β-CateninLeads to HSC Expansion with Loss of Early Hematopoietic ProgenitorsAnimals

All mice used in this study were housed in the animal facility atStowers Institute for Medical Research (SIMR) and handled according toInstitute and NIH guidelines. All procedures were approved by the IACUCof SIMR. Pten/constitutively active β-catenin double mutant mice wereinduced by intra-peritoneal injection of Tamoxifen (Sigma, Cat. No.T5648) everyday for 5 days using 5 mg on day 1 and 2 mg on days 2-5 eachdissolved in 0.1 ml of corn oil (Sigma, Cat. No. C8267) (completedissolution was achieved by 42° C. water bath sonication for about 5minutes). Mx-1 Cre induction was achieved by 250 μg injection of polyl:Cevery other day utilizing 1 dose (for the Mx-1 Cre Pten:β-cat^(Act)model) or 5 doses (for the Mx1-Cre Pten:β-cat^(−/−) transplant model).Scl-Cre, Pten, β-cat^(Act), and β-cat^(−/−) mice were obtained fromJoachim Goethert (University of Duisburg-Essen, Germany), Hong Wu (UCLA,Los Angeles, Calif.), Makoto Taketo (Kyoto University, Japan) and theJackson Laboratory (Bar Harbor, Me.), respectively.

Histology

Paraffin sections of spleen, tumors or decalcified femurs were stainedwith H&E or Masson's Trichrome as indicated.

Immunofluorescent Assays

GFP⁺ HSCs were sorted and transplanted as previously reported (Xie, Y.et al. Detection of functional haematopoietic stem cell niche usingreal-time imaging. Nature 457, 97-101 (2009)). Femurs and tibias werefixed in 4% PFA or Zn²⁺-Formalin and processed for paraffin and frozensections, respectively. For immunofluorescent staining, after antigenretrieval using EZ Retriever Microwave (BioGenex, San Ramon, Calif.) for10 minutes at 95° C. in citrate buffer, non-specific antibody bindingwas blocked by incubating slides with 1× Universal Block (BioGenex,#HK085-5k) at room temperature for 1 hour. pβ-cat-S552 was stained aspreviously reported (He, X. C. et al. PTEN-deficient intestinal stemcells initiate intestinal polyposis. Nat Genet 39, 189-198 (2007)).

Induction of PTEN/Constitutively Active β-Catenin Double Mutant Mice

The inventors have previously demonstrated that PTEN deficiency leads toexcessive intestinal stem cell (ISC) proliferation resulting inintestinal polyposis, a pre-cancerous neoplasia (He, X. C. et al.PTEN-deficient intestinal stem cells initiate intestinal polyposis. NatGenet 39, 189-198 (2007)). Akt has been shown to phosphorylate β-cateninat serine residue 552 (S552), with the resulting phosphorylated form ofβ-catenin being nuclear localized in ISCs. An antibody highly-specificfor β-catenin phosphorylated at S552 (β-cat-pS552) reveals that cellswith nuclear (activated) β-cat-pS552 initiate ISC expansion, resultingin polyposis in PTEN-deficient mice. Staining with β-cat-pS552 antibodyshows simultaneous activation of the two pathways. Considering its rolein ISCs, it was hypothesized that β-cat-pS552 antibody may alsorecognize activated HSCs. To investigate this, purified HSCs whichexpress green fluorescent protein (GFP⁺ HSCs) was transplanted intoirradiated and non-irradiated mice. The recipients were sacrificed, andtheir bone sections were stained with anti-β-cat-p5552 antibody. Withirradiation, a condition previously shown to result in rapid HSCexpansion (Xie, Y. et al. Detection of functional haematopoietic stemcell niche using real-time imaging. Nature 457, 97-101 (2009)), purifiedGFP⁺-HSCs were observed to be adjacent to their endosteal niche with 5of 40 GFP⁺ HSCs costaining as β-cat-pS552⁺ cells, some of which were inthe process of active division (FIGS. 8A-D). However, withoutirradiation, a condition where HSCs do not expand (Id.), 0 of 15 GFP⁺HSCs were found to be β-cat-pS552⁺ (data not shown). β-cat-pS552antibody were also used to visualize Wnt/β-catenin and PTEN/Aktsignaling pathway interaction in control, single, and double mutantspleen. At 3 days post-induction (dpi), control and single mutantspleens showed only rare and lightly stained cells, while double mutantsexhibited more intense and abundant β-cat-pS552 staining (FIG. 14).These results support the importance of activated Akt and β-catenininteraction in normal but proliferating HSCs and show that this pathwayinteraction is enhanced in double mutants compared to single mutants orcontrol.

The consequences of combining both conditional Pten deletion withconstitutive activation of β-catenin (β-cat^(Act)) (Ctnnb1^(tm1Mmt))(Harada, N. et al. Intestinal polyposis in mice with a dominant stablemutation of the beta-catenin gene. The EMBO journal 18, 5931-5942(1999)) was studied using Mx1-Cre. This interferon-inducible systemresults in tissue non-specific knockout of LoxP-flanked (floxed) allelesand has been used in previous studies focusing on either Pten orβ-cat^(Act) single mutants (Kirstetter, P., Anderson, K., Porse, B. T.,Jacobsen, S. E. & Nerlov, C. Activation of the canonical Wnt pathwayleads to loss of hematopoietic stem cell repopulation and multilineagedifferentiation block. Nat Immunol 7, 1048-1056 (2006); Scheller, M. etal. Hematopoietic stem cell and multilineage defects generated byconstitutive [beta]-catenin activation. Nature Immunology 7, 1037-1047(2006); Yilmaz, O. H. et al. Pten dependence distinguisheshaematopoietic stem cells from leukaemia-initiating cells. Nature 441,475-482 (2006); Zhang, J. et al. PTEN maintains haematopoietic stemcells and acts in lineage choice and leukaemia prevention. Nature 441,518-522 (2006)). Mx1-Cre⁺ Pten:β-cat^(Act) double mutants exhibit severehair follicle tumors known as trichofolliculomas that consist ofwell-formed but densely packed hair follicles which rapidly cover thebody (FIG. 15) (Gray, H. R. & Helwig, E. B. Trichofolliculoma. ArchDermatol. 86, 99-105 (1962)).

Because trichofolliculomas made it impossible to complete long-termstudies in these double mutants using Mx1-Cre, Pten and β-cat^(Act)(Ctnnb1^(fl/fl)) single and double mutants were crossed with thetamoxifen-inducible HSC-SCL-Cre-ER^(T) strain (hereafter referred to asScl-Cre), which allowed for studying the effects initiating primarilyfrom HSCs (Gothert, J. R., et al., “In vivo fate-tracing studies usingthe Scl stem cell enhancer: embryonic hematopoietic stem cellssignificantly contribute to adult hematopoiesis.” Blood, 2005. 105(7):p. 2724-2732). The cross is set forth in more detail below.

Mice with homozygous floxed (fl) alleles of Pten (Pten^(fl/fl)) werebred with Ctnnb1^(fl/fl) mice in which exon 3 of the mouse β-cateningene (where all phosphorylation target serine/threonine residues arelocated) was sandwiched by two IoxP sequences. (Harada, N., et al., EmboJ, 18(21): 5931-42 1999. Yilmaz, O. H., et al., Nature, 441:475-82 2006.Zhang, J., et al., Nature, 441(7092): 518-22 2006.) Double heterozygousmice from this cross were then crossed to generate Pten^(fl/fl)Ctnnb1^(fl/+) mice (since Ctnnb1 is a gain-of-function allele, onlyheterozygous mice for Ctnnb1 are necessary). Concurrently, Pten^(fl/fl)mice were bred with Scl-Cre⁺ transgenic mice to generate Scl-Cre⁺Pten^(fl/+) mice. These were then crossed to generate Scl-Cre⁺Pten^(fl/fl) mice (“Pten”). Finally, Pten^(fl/fl) Ctnnb1^(fl/+) micewere bred with Scl-Cre⁺Pten^(fl/fl) mice to generate Scl-Cre⁺Pten^(fl/fl) Ctnnb^(fl/+) mice (“Pten:Ctnnb1”). Scl-Cre mice were alsobred with Ctnnb1^(fl/fl) mice to generate the single mutant Scl-Cre⁺Ctnnb1^(fl/+) mice (“Ctnnb1”). Mice lacking Scl-Cre (“Scl-Cre negative”or “Control”) were used as controls.

HSC Analysis

For phenotype analysis, hematopoietic cells were harvested from bonemarrow (femur and tibia), spleen, peripheral blood, and thymus. Redblood cell lysis was performed using hemolysis buffer (0.16M ammoniumchloride, Sigma Cat. No. A9434). Cells were stained for lineage markersusing CD3, CD4, CD8, B220, IgM, Mac-1, Gr1, and Ter119 antibodies alongwith Kit, and Sca-1 for LSK analysis or these markers along with IL-7Rα,CD34 and CD16/32 for progenitor analysis (Akashi, K., et al., Aclonogenic common myeloid progenitor that gives rise to all myeloidlineages. Nature 2000. 404(6774): p. 193-7). Flk2 was added as indicatedfor LT-HSC analysis.

Unless otherwise indicated, all antibodies were obtained fromeBiosciences (San Diego, Calif.) as indicated below: Fluoresceinisothiocyanate (FITC) conjugated CD3 antibody (Cat. No. 11-0452-85),FITC conjugated CD4 antibody (Cat. No. 11-0042-85), FITC conjugated CD8antibody (Cat. No. 11-0081-85), FITC conjugated B220 antibody (Cat. No.11-0452-85), FITC conjugated Ter119 antibody (Cat. No. 11-5921-85), FITCconjugated Mac-1 antibody (Cat. No. 11-0112-85), FITC conjugated Gr1antibody (Cat. No. 11-5931-85), FITC conjugated IgM antibody (Cat. No.11-5790-85), Phycoerythrin (PE) conjugated Sca-1 antibody (Cat. No.12-5981-83), Allophycocyanin (APC) conjugated Kit antibody (Cat. No.17-1171-83), Biotin conjugated CD135 (Flk-2) antibody (Cat. No.13-1351-85), PE-Cy5 conjugated CD127 (IL-7Rα) antibody (Cat. No.15-1271-83), PE-Cy7 conjugated CD16/32 (FcγRII/III) antibody (Cat. No.25-0161-82), Biotin conjugated CD34 antibody (Cat. No. 13-0341-85),Streptavidin conjugated PE-Cy7 antibody (Cat. No. 25-4317-82),Streptavidin conjugated APC-Cy7 antibody (Cat. No. 10-4317-82), APCconjugated Gr1 antibody

(Cat. No. 17-5931-82), APC conjugated 8220 antibody (Cat. No.17-0452-83), PE conjugated Mac-1 antibody (Cat. No. 12-0112-83), and PEconjugated CD3 antibody (Cat. No. 12-0031-85).

Antibody stained cells were sorted by FACS using a MoFlo (Dako, Ft.Collins, Colo.) flow cytometer and/or a CyAn ADP (Dako, Ft. Collins,Colo.), and analyzed for lineage negative, Sca-1⁺Kit⁺ (LSK) cells inScl-Cre negative control and Scl-Cre⁺ PTEN with constitutively activatedβ-catenin (Pten:Ctnnb1) double mutant bone marrow and spleen. Dataanalysis was performed using FlowJo software (Ashland, Oreg.).

In order to study the consequences of Pten deletion combined withβ-catenin activation on stem cells in vivo, HSCs and early progenitorswere analyzed from single and double mutants bred onto the Scl-Cre line.At 10 days post-induction (dpi), Pten:β-cat^(Act) (hereafter mutants areScl-Cre⁺ unless otherwise specified as Mx1-Cre⁺) LSK cells were slightlyreduced in bone marrow but significantly increased in spleen (p<0.001),suggesting a mobilization of HSCs. Similar to previous reports (Yilmaz,O. H. et al. Pten dependence distinguishes haematopoietic stem cellsfrom leukaemia-initiating cells. Nature 441, 475-482 (2006); Zhang, J.et al. PTEN maintains haematopoietic stem cells and acts in lineagechoice and leukaemia prevention. Nature 441, 518-522 (2006)), LSK cellsin Pten mutants were also significantly increased in spleen (p<0.05),though this expansion was not as great as in double mutant spleen (FIG.1A). At 4 weeks post-induction (wpi), early myeloid progenitorsincluding common myeloid, megakaryocyte-erythroid, andgranulocyte-monocyte progenitors (CMPs, MEPs, and GMPs, respectively)(Akashi, K., Traver, D., Miyamoto, T. & Weissman, I. L. A clonogeniccommon myeloid progenitor that gives rise to all myeloid lineages.Nature 404, 193-197 (2000)) were significantly reduced in Pten:Ctnnb1bone marrow. No other dramatic differences were observed betweencontrol, single, and double mutant bone marrow and spleen (data notshown). By 6 wpi, the frequency of LSK cells increased in Pten:Ctnnb1bone marrow though the absolute number was not significantly increaseddue to low cellularity (FIGS. 1B-C and 1F; cells were pre-gated on live,lineage negative cells.). Strikingly, LSK cells in spleen, whichincreased only modestly in Pten single mutants, were dramaticallyincreased in double mutants (FIGS. 1D-E and 1G). The LSK population wasfurther subdivided based on Flk2 expression, which allowed for furtherenrichment long-term reconstituting (LT) HSCs (F1k2⁻) from short-termreconstituting (ST) HSCs (Flk2⁺) (Christensen, J. L. & Weissman, I. L.Flk-2 is a marker in hematopoietic stem cell differentiation: a simplemethod to isolate long-term stem cells. Proceedings of the NationalAcademy of Sciences of the United States of America 98, 14541-14546(2001)). Compared to control and single mutants, a significantly higherpercentage of LSK cells from double mutants were Flk2⁻, suggesting thatthe expansion occurred predominantly in the LT-HSC subpopulation (FIG.1H-I). By 6 wpi, about 50% of double mutants began to develop leukemiawith substantial blast cell (CD45^(High)) populations (FIG. 1J and datanot shown) (Borowitz, M. J., Guenther, K. L., Shults, K. E., Stelzer, G.T. Immunophenotyping of acute leukemia by flow cytometric analysis. Am.J. Clin. Pathol. 100, 534-540 (1993)). As shown in FIG. 1J, CD45 (high)blast crisis cells are indicated in the blue box of the left panel. LSKanalysis of leukemic Pten:Ctnnb1 mutant mouse bone marrow was alsoperformed (FIG. 1J, right panel). Note the conversion to blast cells(lower left) with only a remnant LSK population (compare to FIG. 1C).These leukemic mice were excluded from the analyses presented in therest of FIG. 1 because their LSK population was reduced when blast cellsincreased and out-competed other cells, which was accompanied bystromal/niche disruption (see below). In comparison, no blast cellpopulation is observed in control or Ctnnb1 single mutants while a minorone was observed in 1 of 8 Pten single mutant mice at 6 weekspost-induction (data not shown).

Early hematopoietic progenitors were also analyzed in control, single,and double mutants. Notably, while LSK populations were increased priorto leukemia development in double mutants, the Lin⁻, Sca-1⁻, Kit⁺population which contained early myeloid progenitors was reduced (FIGS.1B-E) (Akashi, K., Traver, D., Miyamoto, T. & Weissman, I. L. Aclonogenic common myeloid progenitor that gives rise to all myeloidlineages. Nature 404, 193-197 (2000)). Absolute numbers of these earlyprogenitors including CMPs, GMPs, and MEPs were substantially reduced indouble but not single mutants, while the common lymphoid progenitor(CLP) number (Kondo, M., Weissman, I. L. & Akashi, K. Identification ofclonogenic common lymphoid progenitors in mouse bone marrow. Cell 91,661-672 (1997)) was similar to control (FIG. 1K), despite the doublemutant's having substantially more HSCs (FIGS. 1E, 1G, and 1I). Thesedata demonstrate that unlike either single mutant, double mutantsexhibit a dramatic expansion in LT-HSCs with differentiation suppressionof myeloid lineages but without corresponding increases in lymphoiddifferentiation. In contrast, Pten single mutants exhibit excessivemyeloid differentiation, progressing to MPD (FIG. 9).

Together, this data demonstrates the phenotypic effect of the geneticloss of PTEN coupled with constitutive activation of β-catenin in HSCs.While loss of PTEN alone results in a slight but significant expansionin splenic HSCs due to mobilization from the bone marrow, double mutantHSCs exhibit the greatest mobilization at 10 days post-induction. By sixweeks post-induction, only double mutant splenic HSCs are dramaticallyincreased while single mutants are not significantly different fromcontrols. In addition, this dramatic increase in HSCs is not accompaniedby an increase in early hematopoietic progenitors; rather these earlyprogenitors are all reduced with the exception of CLPs which are notsignificantly different from control. HSCs accumulate dramatically inthe spleen of double, but not single, mutants by proliferation withreduced differentiation. Thus, surprisingly and unexpectedly, loss ofPTEN coupled with the constitutive activation of β-catenin drives stemcell self-renewal while neither pathway individually is capable ofdriving long-term self-renewal.

Example 2 In Vitro Culture of Control and Mutant LSK Cells Cell Culture

LSK or LSK Flk2⁻ cells were sorted into 96-well U-bottom tissue cultureplates at 100 cells/well with 200 μl media/well. Cells were incubated at37° C., 5% O₂, 5% CO₂ (balance N₂) for the indicated number of days.One-half total volume of media (see Table 1, below for the base media)was carefully pipetted from the top and replaced with fresh media everyother day.

TABLE 1 Base Media Components Source StemSpan Media: (Iscove's-modifiedDulbecco's Stem Cell medium (IMDM) supplemented with 1% bovineTechnologies; serum albumin, 10 μg ml⁻¹ recombinant human Cat. No. 09600insulin, 200 μg ml⁻¹ iron-saturated transferrin, 0.1 mM2-mercaptoethanol and 2 mM glutamine.) 10 μg/ml Heparin Sigma, Cat. No.H-3149 0.5X Penicillin/Streptomycin Sigma, Cat. No. P4333 10 ng/mlrecombinant mouse (rm) Stem Cell Biovision, Cat. Factor No. 4328-10 20ng/ml rm-Thrombopoietin Cell Sciences, Inc, Cat. No. CRT401B

Double Mutant HSCs Expand Dramatically In Vitro and In Vivo but Fail toDifferentiate.

For the following experiments, the base media from Table 1 was furthersupplemented with 20 ng/ml rm-IGF-2 (R&D Systems, Cat. No. 792-MG) and10 ng/ml recombinant human FGF-1 (Affinity BioReagents, Cat. No.ORP16010).

The ability of HSCs isolated from Mx1-Cre⁺ single and double mutants toexpand in vitro was examined. Lineage negative, Sca-1⁺, Kit⁺ (LSK) cells(a population highly enriched in HSCs) were sorted from wild-type(control), single, and double mutant Mx1-Cre⁺ bone marrow and culturedin defined media based on a previous report regarding ex vivo HSCexpansion (Zhang, C. C. & Lodish, H. F. Murine hematopoietic stem cellschange their surface phenotype during ex vivo expansion. Blood 105,4314-4320 (2005)). After 10 days culture, control LSK cells hadundergone a modest expansion; however, Ctnnb1 LSK cells did not survive,suggesting they had undergone apoptosis. In contrast, Pten LSK culturesexpanded to a greater degree than control, while the best expansion wasobserved from double mutant cultures (FIG. 2A). Pten and Pten:Ctnnb1cultures continued to expand up to 5 weeks in vitro (FIG. 2B); however,control cultures began to decline after 4 weeks (data not shown). Unlikecontrol, both Pten and Pten:Ctnnb1 cultures remained robust after 5weeks, but Pten:Ctnnb1 cultures contained far more cells and theirappearance was more homogenous than Pten cultures (FIG. 2B). At 7 weeks,a portion of the remaining Pten and Pten:Ctnnb1 cultures was re-analyzedby fluorescence-activated cell scanning (FACS) analysis to determine howmany cells had maintained their LSK phenotype (FIG. 2E). While LSK cellsfrom Pten cultures had expanded 50-fold, Pten:Ctnnb1 cultures expandedmore than 1,200-fold (FIG. 2C). In addition, the purity of LSK cells (%of total cells maintaining the LSK phenotype) was significantly higherin Pten:Ctnnb1 cultures compared to Pten only (84% vs. 52%,respectively, FIG. 2D).

Example 3 Transplantation Analysis of Pten and Pten:Ctnnb1 LSK CellsAfter 5 Weeks of Culture

For the following experiments, cells were cultured in the same manner asdescribed in Example 2. As in Example 2, the base media of Table 1 wassupplemented with 20 ng/ml rm-IGF-2 (R&D Systems, 792-MG) and 10 ng/mlrecombinant human FGF-1 (Affinity BioReagents, ORP16010).

While Pten and especially Pten:Ctnnb1 cultures exhibited significantexpansion in LSK cells, whether these cells were functional in vivo wasdetermined.

At 5 weeks culture, Pten and Pten:Ctnnb1 LSK cultures were re-sorted and1000 LSK cells (CD45.2⁺) from each were transplanted into lethallyirradiated (10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic wholebone marrow competitor cells. Because wild-type cells did not survive 5weeks culture, 1000 fresh wild-type LSK cells were also transplanted asa separate control group. Peripheral blood analysis at 4 weekspost-transplantation revealed robust repopulation in mice transplantedwith fresh/uncultured control cells as expected; however, micetransplanted with either Pten or Pten:Ctnnb1 cultured cells did notexhibit repopulation (data not shown). At 5 weeks post-transplant, bonemarrow from recipient mice was analyzed for donor engraftment (CD45.2⁺cells) and donor LSK cells (CD45.2⁺ LSKs).

To determine whether LSK or other donor-derived (CD45.2+) cells remainedin the bone marrow of mice transplanted with cultured cells, bone marrowwas analyzed for donor (CD45.2+) and LSK cells. While the control groupexhibited robust repopulation of CD45.2+ bone marrow cells, few CD45.2+cells were maintained as LSK cells as expected (FIGS. 2C-D and 2F). Incontrast, recipients transplanted with in vitro expanded Pten orPten:Ctnnb1 mutant LSK cells exhibited few donor-derived total bonemarrow cells (FIGS. 2G-H). However, a large portion of Pten:Ctnnb1donor-derived cells were maintained as LSK cells in recipients, whereasthose from Pten only cultures were similar in number to control (FIGS.2G-I). In order to determine whether ex vivo expanded donor cells hadfurther expanded in vivo following transplantation, the total number ofdonor LSK cells in total bone marrow per mouse were estimated (Smith, L.H. & Clayton, M. L. Distribution of injected 59Fe in mice. Exp. Hematol.20, 82-86 (1970)). As shown in FIG. 2J, the expansion of totaldonor-derived LSK cells in transplant recipients was modest and similarbetween control and Pten (8.6±1.4 and 13±6.3, respectively), butsignificantly greater in recipients transplanted with culturedPten:Ctnnb1 LSK cells (43±3.4).

Collectively, these data demonstrate that double mutant HSCs can becultured longer and with far greater expansion than either single mutantor control HSCs. However, permanent genetic alteration of both pathwaysleads to an increased ability to self-renew both in vitro as well as invivo following long-term culture but a failure to differentiate and thusrepopulate the hematopoietic system of transplant recipients. Thisfurther demonstrates the ability of the PTEN and β-catenin signalingpathways to cooperatively drive stem cell expansion by proliferationwithout differentiation.

Example 4 Differentiation Block and Dominant Phenotype of Pten:Ctnnb1Mutant HSCs

Initially, primary (non-transplanted) animals were used for phenotypicanalysis. These mice eventually exhibited severe non-hematopoieticdefects, including reduction of the marrow cavity and splenic fibrosisresulting in disruption of splenic niches (FIG. 16). Consequently,LT-HSC transplantations were used to verify Scl-Cre specificity (FIG.10). Comparing these transplant groups with the initial data fromprimary mutants revealed an essentially identical phenotypicmanifestation of defects between transplant and non-transplant groups,demonstrating that non-hematopoietic effects are due to interactionbetween the hematopoietic system and stroma rather than from defectsarising from the stroma (FIG. 17 and data not shown).

The health of double mutants typically declined by 9 wpi (see below).LSK cells and early progenitors from control, single, and double mutantbone marrow and spleen at 9-10 wpi were analyzed by FACS (FIGS. 10A-B).Absolute number of LSK cells were reduced in bone marrow and spleen ofCtnnb1 single mutants but increased in the spleens of Pten singlemutants (FIGS. 10A and 10C-D). CMPs and MEPs were increased in Pten bonemarrow and spleen (FIG. 10B-D). In contrast, LSK cells and all earlyprogenitors including CMPs, MEPs, GMPs and CLPs (Akashi, K., Traver, D.,Miyamoto, T. & Weissman, I. L. A clonogenic common myeloid progenitorthat gives rise to all myeloid lineages. Nature 404, 193-197 (2000);Kondo, M., Weissman, I. L. & Akashi, K. Identification of clonogeniccommon lymphoid progenitors in mouse bone marrow. Cell 91, 661-672(1997)) were severely depleted in Pten:Ctnnb1 bone marrow.Interestingly, when leukemic cells predominated but health had not yetseverely declined, Pten:Ctnnb1 mutants exhibited a distinct populationof Lin− Sca-1^(Low) Kit^(Mid) (LS^(Low)K^(Mid)) cells at 9 wpi (FIG.10A, panels IV and IX). At 10 wpi when health had severely declined,this population was typically absent and only leukemic blast cellsremained (FIG. 10A, panels V and X).

CD45.1 (recipient) and CD45.2 (donor) markers were used to measureengraftment levels in recipients at 9-10 wpi. As expected, robustengraftment was observed in recipients of 1,000 control LSK Flk2− cells(77±6%) (FIG. 10E). Pten mutants exhibited somewhat higher averageengraftment of 88±4%. The highest and most consistent engraftment of97±1.5% was exhibited in Pten:Ctnnb1 mice. In contrast, averageengraftment was only 32±36% in Ctnnb1 mutants, with half the recipientsexhibiting little to no engraftment. The relatively poor and variableengraftment observed in the Ctnnb1 transplant group may be due to aminor portion of HSCs that escaped knockout of the floxed Ctnnb1 allele.Indeed, previous reports have shown that phenotypically defined HSCs inCtnnb1 mutants are no longer functional (Kirstetter, P., Anderson, K.,Porse, B. T., Jacobsen, S. E. & Nerlov, C. Activation of the canonicalWnt pathway leads to loss of hematopoietic stem cell repopulation andmultilineage differentiation block. Nat Immunol 7, 1048-1056 (2006);Scheller, M. et al. Hematopoietic stem cell and multilineage defectsgenerated by constitutive [bet]-catenin activation. Nature Immunology 7,1037-1047 (2006)). In order to test this, LSK Flk2− cells from Scl-Crenegative (control) as well as Ctnnb1 mutants at 2 and 16 wpi were sortedand genotyped for presence of the knockout allele. At 2 wpi, the mutantCtnnb1 allele was present; however, by 16 wpi no cells containing mutantCtnnb1 allele remained, demonstrating that Ctnnb1 mutant HSCs are notmaintained long-term (FIG. 18). In contrast, Pten:Ctnnb1 HSCs werehighly dominant and almost wholly out-competed all HSCs found within thecompetitor bone marrow cells. To verify this, the Z/EG reporter systemwere included to determine which cells had undergone Cre-mediatedexcision of their floxed alleles (Novak, A., Guo, C., Yang, W., Nagy, A.& Lobe, C. G. Z/EG, a double reporter mouse line that expresses enhancedgreen fluorescent protein upon Cre-mediated excision. Genesis 28,147-155 (2000)). The Z/EG reporter system activates expression ofenhanced green fluorescent protein (EGFP) upon Cre-mediated excision. Asexpected, mice transplanted with Scl-Cre negative (control)-Z/EG donorLSK Flk2- cells exhibited no EGFP⁺ LSK Flk2⁻ cells (0.8±0.8%) (FIG.10F). Similarly, recipients of Ctnnb1-Z/EG LSK Flk2⁻ cells also had veryfew EGFP+ LSK Flk2− (1.6±0.9%) cells, further demonstrating thatessentially only those LSK Flk2− cells escaping knockout inductionremained. LSK Flk2⁻ cells from Pten-Z/EG transplant recipients exhibiteda minor GFP⁺ population (8.6±2.1%), demonstrating that some mutant LSKFlk2⁻ cells remained even after 9-10 wpi, although most haddifferentiated or had been otherwise lost. In contrast, LSK Flk2⁻ cellsfrom Pten:Ctnnb1-Z/EG transplant recipients were 90.0±4.0% EGFP+.

Because the exact time point when Ctnnb1 HSCs were lost in vivo is notknow, whether Ctnnb1 HSCs undergo apoptosis was tested. LSK Flk2⁻ cellswere isolated from uninduced mice, genetic deletion was induced invitro, and the resulting cultures were then visually monitored. Theseexperiments revealed that, by 4 days post-induction, no Ctnnb1 LSK Flk2⁻cells survived; whereas control, Pten, and particularly double mutantLSK Flk2⁻ cells survived and expanded (FIG. 11A). At 48 hourspost-induction, although some Ctnnb1 LSK Flk2⁻ cells remained, theirnumbers were reduced relative to control (FIG. 11B). Whether these cellswere undergoing apoptosis was tested by Annexin V staining. Unlikecontrol, the majority of Ctnnb1 LSK Flk2⁻ cells at hours post-inductionwere either undergoing apoptosis or already dead, demonstrating thatconstitutive activation of β-catenin in LSK Flk2⁻ cells in vitro resultsin rapid apoptosis (FIG. 11C). These data demonstrate that while mostPten mutant HSCs differentiate, Ctnnb1 mutant HSCs undergo rapidapoptosis in vitro, exhibit functional failure in vivo, and are notmaintained in recipients. In contrast, double mutant LT-NCSs werephenotypically maintained 9-10 wpi, becoming the dominant HSC populationin transplant recipients.

Example 5 Loss-of-Function β-Catenin Prevents PTEN-Deficiency-InducedHSC Expansion But Not MPD Colony Forming Unit (CFU) Assays

CFU Assays were performed according to manufacturer's instructions usingcomplete methylcellulose media with Epo (Cat. No. M3434, Stem CellTechnologies, Inc., Vancouver, Canada).

Lentiviral Production/HSC Transduction

To knockdown mouse β-catenin in HSCs and their progeny, lentiviruses inwhich mouse β-catenin short-hairpin RNAs (shRNAs) and an IRES GFP labelare driven by a MSCV (murine stem cell virus) LTR promoter weregenerated. Lentiviral constructs were produced by directionally cloningDNA oligonucleotides corresponding to two Ctnnb1 shRNAs (sequencesHP_(—)224742 and HP_(—)240000; SEQ ID NOs: 3-4, RNAicodex, Cold Spring,N.Y.) into the Gateway® entry vector pEN-LmiRc3 (Invitrogen), thenrecombining with the destination vector pDSL-hplG (Zhu, X. et al. Aversatile approach to multiple gene RNA interference usingmicroRNA-based short hairpin RNAs. BMC molecular biology 8, 98 (2007)).The control viral construct drives a luciferase shRNA (Id.) from thesame vector backbone. Virus was produced in 293T cells byco-transfection of the virus plasmid with packaging plasmids(pRC-CMV-Rall, HDM-Tat16, HDM-HGPM2 and HDM-VSVG, a gift from Dr.Jeffrey M. Rosen, Baylor College of Medicine); and was purified bypoly-ethylene-glycol (PEG) precipitation (0.45 μm-filtered supernatantwas precipitated with 10% PEG-8000, 1.5% fetal bovine serum for 72 hoursat 4° C., then pelleted at 1,500 g for 10 minutes), followed byultracentrifugation through a sucrose cushion (al Yacoub, N.,Romanowska, M., Haritonova, N. & Foerster, J. Optimized production andconcentration of lentiviral vectors containing large inserts. Thejournal of gene medicine 9, 579-584 (2007)). Titres were establishedusing 293T cells due to the limited numbers of HSCs available. Viraltransduction of HSCs was performed overnight in ST media with 8 μM finalpolybrene at a multiplicity of infection of 20-50 relative to theinitial cell HSC number (500 cells).

Transplantation Assays

For the β-cat^(−/−) experiments, whole bone marrow transplants wereperformed into lethally irradiated Ptprc recipients using 1×10⁶cells/recipient.

Experiment Results

In order to more comprehensively study the role of β-catenin interactionwith the PTEN/Akt signaling pathway, mice with floxed null alleles ofβ-catenin (Ctnnb1^(tm2Kem)) (Cobas, M. et al. Beta-catenin isdispensable for hematopoiesis and lymphopoiesis. The Journal ofexperimental medicine 199, 221-229 (2004)) were obtained and crossed toMx1-Cre and Mx1-Cre Pten mutants, allowing for the combination ofconditional deletion of β-catenin (β-cat^(−/−)), Pten, andPten:β-cat^(−/−). As with the Pten:Ctnnb1 compound mutant, primaryanimals were studied; however, this was difficult to pursue becauseβ-cat^(−/−) mice typically had to be sacrificed by 15 dpi, whilePten:β-cat^(−/−) double mutants rarely maintained adequate health beyond7 dpi (data not shown). In order to study long-term andhematopoietic-specific defects in single and double knockout mutants,whole bone marrow transplantations were performed. Bone marrow fromcontrol (Cre⁻), Mx1-Cre⁺ single and double mutant donors into lethallyirradiated Ptprc recipients prior to induction. At 10 wpi of transplantrecipients, 5 mice from each group were sacrificed, and LSK cells aswell as early progenitors were analyzed by FACS. Unlike Pten:Ctnnb1double mutants, none of the Pten:β-cat^(−/−) double mutants exhibitedsigns of leukemia by 10 wpi (data not shown). Consistent with previousreports, β-cat^(−/−) single mutants did not exhibit any defects inabsolute numbers of LSK or early progenitors (Cobas, M. et al.Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. TheJournal of experimental medicine 199, 221-229 (2004)); however Ptensingle mutants exhibited an expansion of LSK cells as well as CMPs andMEPs in the spleen (FIG. 11D). Interestingly, Pten:β-cat^(−/−) doubleknockout transplant recipients did not exhibit an expansion of LSK cellsin the spleen, while CMPs and MEPs were increased compared to controlbut less than Pten only mutants (FIG. 11D). In contrast, analysis ofmore mature hematopoietic lineages revealed similar increases in Mac-1⁺Gr1⁺ cells between Pten single and Pten:β-cat^(−/−) double knockouts,indicative of MPD, while lymphoid lineages were similarly reduced inboth Pten and Pten:β-cat^(−/−) transplant recipients (FIGS. 11E-G).These results demonstrate that loss of β-catenin rescues the LSK cellexpansion observed in Pten mutant spleen and partially rescues the earlymyeloid progenitor cell expansion, although MPD development stilloccurs. Relative to the number of LSK cells, Pten:β-cat^(−/−) mutantsexpanded early myeloid progenitors as well as or greater than Ptensingle mutants (FIG. 11D). Thus, loss of β-catenin appears to primarilyrescue HSC-specific effects, with the downstream events that lead to MPDbeing separable, β-catenin-independent phenomena. These data alsofurther confirm that the Wnt/β-catenin and PTEN/Akt pathwayscooperatively interact in driving HSC expansion.

Although putative HSCs can be highly enriched by cell surface markerphenotype, bona fide HSCs are functionally defined. When geneticmutation compromises function, formal proof that a putative HSCpopulation represents true HSCs can be precluded. This is the case formutants with constitutively active β-catenin because differentiation isblocked. Whether LSK Flk2⁻ cells isolated from double mutants couldrecover multilineage differentiation capacity if β-catenin transcriptswere degraded by RNA interference (RNAi) were determined. LSK Flk2⁻cells from uninduced control, Pten, and Pten:Ctnnb1 mice were sorted andinduced knockout in vitro with 4-hydroxy-tamoxifen (OHT) added for 3days in culture. At day 3, HSC cultures were transduced using lentiviralvectors targeting β-catenin transcripts by RNAi as set forth below.

At day 6, colony forming unit (CFU) assays were performed on these HSCcultures. While knockdown of β-catenin in control and Pten HSC culturesdid not significantly affect colony formation, knockdown of β-catenin indouble mutant cultures resulted in reversal from a novel CFU phenotypeto a CFU phenotype similar to Pten single mutants (FIGS. 19A-B).Specifically, double mutant HSC cultures transduced with control vectorformed large CFU (>0.5 mm) which were not produced in control or Ptencultures. -Interestingly, these primitive CFU were mostly CD3⁺ (Tlymphoid) cells not found in control colonies (FIG. 19C). Double mutantcultures transduced with short-hairpin (sh) RNA targeting β-cateninproduced only small CFU similar to control and Pten. These smallercolonies further contained only minor proportions of CD3⁺ cells. Inaddition, the number of colonies was shifted toward a predominance ofgranulocyte/monocyte progenitors (CFU-GM) similar to Pten single mutants(FIGS. 19A-C). These data demonstrate that the differentiation blockageexhibited by double mutant LSK Flk2⁻ cells is functionally reversible,supporting the idea that the phenotypically defined HSCs expanding indouble mutants are, indeed, bona fide, though functionally compromised,HSCs.

Example 6 Unlike Single Mutants, Double Mutants Rapidly and ConsistentlyDevelop Leukemia

Control animals (Scl-Cre negative littermates) remained healthy asexpected, and Ctnnb1 mutants also remained healthy through at least 20wpi. In contrast, about 30% of Pten single mutants had to be sacrificedby 20 wpi, but the majority survived through at least 28 wpi.Pten:Ctnnb1 double mutants exhibited a far more rapid decline in healththan Pten single mutants. Double mutants typically survived until atleast 8 wpi when a minority had to be sacrificed due to poor condition(FIG. 12A). By 11 wpi, however, all double mutants had to be sacrificed.Histological examination of Pten:Ctnnb1 bone marrow at 9-10 wpi revealedthat the bone shaft (diaphysis) became substantially filled with bone,while trabecular bone regions (metaphysis), reported to be enriched insites containing the HSC niche (Xie, Y. et al. Detection of functionalhaematopoietic stem cell niche using real-time imaging. Nature 457,97-101 (2009); Arai, F. et al. Tie2/angiopoietin-1 signaling regulateshematopoietic stem cell quiescence in the bone marrow niche. Cell 118,149-161 (2004); Calvi, L. M. et al. Osteoblastic cells regulate thehaematopoietic stem cell niche. Nature 425, 841-846 (2003); Zhang, J. etal. Identification of the haematopoietic stem cell niche and control ofthe niche size. Nature 425, 836-841 (2003)), were largely hypo-cellularwith areas that appeared grossly normal (FIG. 11B). In contrast, noobvious defects were apparent in either single mutant bone marrow (datanot shown). Splenomegaly in Pten:Ctnnb1 mutants at 9-10 wpi wasobserved, with the spleen exhibiting severe hypo-cellularity andfibrosis (FIG. 16). In contrast, gross appearance of single mutantspleen was normal at 9-10 wpi. Furthermore, the hypo-cellularity andfibrosis observed in double mutant spleen was present even in wild-typemice transplanted with LSK Flk2-cells from Pten:Ctnnb1 donors. Thestromal abnormalities observed were most likely a consequence of loss ofnegative inhibition from hematopoietic cells to normal stroma ratherthan of defects originating in the stroma.

Suspecting that acute leukemia/lymphoma caused the rapid decline inhealth of Pten:Ctnnb1 mutants between 8-11 wpi, FACS analysis was usedto examine the abundance of CD45^(High) primitive blast cells incontrol, single, and double mutants (Borowitz, M. J., Guenther, K. L.,Shults, K. E., Stelzer, G. T. Immunophenotyping of acute leukemia byflow cytometric analysis. Am. J. Clin. Pathol. 100, 534-540 (1993)). Asshown in FIG. 5C, Pten:Ctnnb1 mutants exhibited a conversion topredominantly leukemic blast cells in the bone marrow by 9-10 wpi. Thiswas observed in all double mutants examined (n>20). In contrast, controland Ctnnb1 mice never exhibited a significant blast population.Typically, Pten mutants were also similar to control regarding bonemarrow CD45 expression level at 9-10 wpi, although 2/16 exhibited aminor blast population (data not shown). These data demonstrate that allPten:Ctnnb1 mutants develop a severe acute leukemia by 9-10 wpi whilesingle mutants do not. Lineage marker analysis further characterized theleukemic cells to express the T-cell specific marker CD3, revealing theleukemia to be T-cell acute lymphocytic leukemia or T-ALL (FIG. 5C).

To further investigate hematopoietic lineage defects in Pten:Ctnnb1mutants and to characterize the type of leukemia, the majorhematopoietic lineages was examined in bone marrow at 8-9 wpi (FIGS.20A-G). Most prominently, CD3⁺ cells in double mutants did not expressmore differentiated T-cell markers, CD4 or CD8. Overall, more than 75%of total bone marrow cells in double mutants were CD3⁺ but CD4 and CD8negative, compared with less than 5% in control (FIGS. 20B-C). Tofurther define the origin and nature of the T-ALL observed in doublemutants, T-cell development in thymus was also examined. Double negative(DN) early T-cell precursors lack CD3, 4 and 8 expression, and theirstage of maturation can be distinguished by CD25 and/or CD44 expression.While less than 5% of thymocytes were within the DN subset in controland single mutants, the majority of thymocytes were within this subsetin double mutants (FIGS. 20D-E). Although both single mutants weresimilar to control, Pten:Ctnnb1 mice exhibited a large increase in DNCD25− CD44− cells (FIGS. 20D-E). Also, while the majority of thymocyteswere double positive precursors in control and single mutants asexpected, this population was essentially absent from Pten:Ctnnb1 mice(FIGS. 20E-G). These data demonstrate that the T-ALL observed in doublemutants involves expansion of an early thymic progenitor, resulting inthe accumulation of immature T-lineage precursors.

Self-renewal has been proposed to require the co-occurrence of threeevents, proliferation while preventing apoptosis and blockingdifferentiation (Zhang, J. & Li, L. BMP signaling and stem cellregulation. Dev Biol 284, 1-11 (2005)). By studying the individual andcombined effects of PTEN and β-cat mutants, the inventors havediscovered that HSC self-renewal is cooperatively controlled by thePTEN/Akt and Wnt/β-catenin pathways acting in a manner consistent withthis tripartite view of self-renewal. Switching from a non-tissuespecific method of gene disruption to generating HSC-specificconditional mutants using the Scl-Cre system allowed for the study ofdefects arising primarily from HSCs and for the long-term, controlledstudy of double mutants. With this more refined model, it was found thatPten deletion results in relatively moderate HSC proliferation andincreased myeloid differentiation. Pten deletion also results in Aktactivation, a potent cell survival factor which prevents apoptosis(Datta, S. R. et al. Akt phosphorylation of BAD couples survival signalsto the cell-intrinsic death machinery. Cell 91, 231-241 (1997); Salmena,L., Carracedo, A. & Pandolfi, P. P. Tenets of PTEN Tumor Suppression.Cell 133, 403-414 (2008)). In contrast, Wnt/β-catenin signaling blocksdifferentiation, but additional signals are needed for HSC expansion.Similarly, all β-catenin and most Pten single mutants fail to developleukemia, which requires aberrant self-renewal. However, theseexperiments demonstrates that only in cooperation can Wnt/β-catenin andPTEN/Akt signaling drive self-renewal and expansion without extensivedifferentiation. Although permanent mutation in both these pathwaysultimately leads to T-ALL, transient, pharmacological manipulationallows for the expansion of functional HSCs. Thus, at the stem celllevel, the interaction between these two pathways coordinates thenecessary components of self-renewal, with each pathway making unique aswell as joint contributions to HSC expansion.

These findings that Pten:Ctnnb1 double mutants expand HSCs to a greaterdegree than single mutants and that compound loss of both β-catenin andPten rescues Pten-deficiency-induced HSC expansion demonstrate that theeffects of Pten loss on HSCs are partially mediated through β-catenin.Rapamycin treatment has been reported to prevent the formation ofleukemia-initiating cells in Pten mutants and to restore normal HSCfunction, indicating that mammalian target of rapamycin (mTor) is alsoan important mediator of the effects of Pten-deficiency (Yilmaz, O. H.et al. Pten dependence distinguishes haematopoietic stem cells fromleukaemia-initiating cells. Nature 441, 475-482 (2006)).

A recent study using VE-cadherin-Cre mediated deletion of Pten hasdemonstrated that leukemic stem cells are highly enriched in arelatively rare population of Kit^(Mid) CD3+ Lin− cells, which appear tobe driven by increased β-catenin activation (Guo, W. et al.Multi-genetic events collaboratively contribute to Pten-null leukaemiastem-cell formation. Nature 453, 529-533 (2008)). Thus, excessiveself-renewal driven by Wnt/β-catenin and PTEN/Akt interaction may beimportant in cancer stem cell development as well as normal HSCself-renewal. Defining the origins and characteristics of cancer stemcells is critical if they are to be detected, if their formation is tobe prevented, or if they are to be selectively eliminated. In theinstant application, the HSC-like LS^(Low)K^(Mid) population frequentlyobserved in double mutants prior to being out-competed by leukemic blastcells (FIG. 10A) may be of particular interest. As a primitivepopulation these could be cancer stem cells or they could be theultimate source of a more mature population of CD3+ cancer stem cells,possibilities that require further testing.

Example 7 Ex Vivo Pharmacological Manipulation of the PTEN/Akt andWnt/β-Catenin Signaling Pathways Cooperatively Drive Functional HSCExpansion

In double mutants, permanent genetic alteration leads to enhancedself-renewal, while differentiation is blocked except toward earlyT-cell commitment, ultimately resulting in T-ALL. The conversion ofessentially all bone marrow cells to competitive leukemic blast cellsalong with the niche disruption prevents sustained HSC expansion indouble mutants. However, reversible, pharmacological manipulation of thePTEN/Akt and/or Wnt/β-catenin pathways may allow for the transientenhancement of self-renewal in vitro with the capacity to function asnormal HSCs following removal of these agents and in vivotransplantation.

This concept was tested by utilizing a small molecule inhibitor of GSK3β(CHIR99021) (Ring, D. B. et al. Selective glycogen synthase kinase 3inhibitors potentiate insulin activation of glucose transport andutilization in vitro and in vivo. Diabetes 52, 588-595 (2003); Schmid,A. C., Byrne, R. D., Vilar, R. & Woscholski, R. Bisperoxovanadiumcompounds are potent PTEN inhibitors. FEBS Lett 566, 35-38 (2004)).GSK3β inhibits β-catenin by targeting β-catenin for proteosomaldegradation and acts in the Wnt/β-catenin pathway. Indeed, CHIR99021,which is the most specific and potent small molecule inhibitor of theWnt/β-catenin pathway reported (Ring, D. B. et al. Selective glycogensynthase kinase 3 inhibitors potentiate insulin activation of glucosetransport and utilization in vitro and in vivo. Diabetes 52, 588-595(2003)), has been shown to promote embryonic stem (ES) cell self-renewaland expansion (Ying, Q.-L. et al. The ground state of embryonic stemcell self-renewal. Nature 453, 519-523 (2008)).

The inventors have developed a defined culture system utilizing only twocytokines, stem cell factor (SCF) and thrombopoietin (Tpo) (ST media),which have been shown previously to support HSC expansion in vitro(Zhang, C. C. & Lodish, H. F. Murine hematopoietic stem cells changetheir surface phenotype during ex vivo expansion. Blood 105, 4314-4320(2005)). While addition of CHIR99021 increased the expansion of LSKFlk2⁻ cells, addition of a small molecule inhibitor of PI3K (NVP-BEZ235)(Maira, S. M. et al. Identification and characterization of NVP-BEZ235,a new orally available dual phosphatidylinositol 3-kinase/mammaliantarget of rapamycin inhibitor with potent in vivo antitumor activity.Molecular cancer therapeutics 7, 1851-1863 (2008)) decreased the abilityof LSK Flk2⁻ cells to expand in a dose-dependent manner (FIG. 21). Also,the ability of CHIR99021 to enhance expansion was negated by PI3Kinhibition (FIG. 21).

One hundred LSK Flk2⁻ cells were sorted from wild-type (C57BI/6) miceand cultured in (1) media, (2) media+1 μM CHIR99021 (a GSK-3β inhibitor,a gift from Dr. Sheng Ding), (3) media+200 nM DipotassiumBis-peroxo(picolinato)oxovanadate (BpV(pic), a PTEN inhibitor, availablefrom Calbiochem, Cat. No. 203705), (4) media+1 μM CHIR99021+200 nMBpV(pic), (5) media+200 nM Shikonin (also a PTEN inhibitor, availablefrom Calbiochem, Cat. No. 565850), and (6) media+200 nM Shikonin+1 μMCHIR99021. (FIGS. 3B-C). Cells were cultured as described above. Cellswere examined at 17 days of culture (FIG. 3B, original magnification100×) and 23 days (FIG. 3C, original magnification 40×). Compared tocontrol, both inhibitors applied individually exhibited greaterexpansion of LSK cells indicating that GSK-3β inhibition is not strictlyequivalent to constitutive activation of β-catenin shown in Ctnnb1mutant LSKs, while BpV(pic) exhibited similar results compared to Ptenmutant LSKs (see FIG. 2). Similar to double mutant LSKs (FIG. 2), thegreatest expansion occurred with both inhibitors present (FIG. 3B/Cpanel 4).

LSK Flk2⁻ cells at 28 days culture in the indicated media conditionswere examined (FIG. 3D, original magnification 200×). Here, significantexpansion relative to control was observed with both inhibitors presentindividually; however, significant differentiation/heterogeneity of cellmorphology was observed in both cases, including more variable cellsize/morphology and/or differentiation to adherent, spindle-shaped cells(middle panels). In contrast, expansion with homogeneity was achievedwhen both inhibitors were present (last panel).

FACS analysis of 28 day LSK Flk2⁻ cells cultured inmedia+BpV(pic)+CHIR99021 (FIG. 3E) was performed. Cells were pre-gatedon live, lineage negative cells. Greater than 90% of LSKs retained Flk2negativity (data not shown). Thus, the LSK Flk2⁻ phenotype wasmaintained with high purity in cultures containing both inhibitors.

Fold expansion of LSK Flk2⁻ cells after 28 days culture in (1) media,(2) media+BpV(pic), (3) media+CHIR99021, and (4)media+CHIR99021+BpV(pic) were analyzed. While each inhibitor addedindividually led to significant expansion compared to media withouteither inhibitor, the greatest expansion (˜270 fold) was observed whenboth inhibitors were added together.

Example 8 Transplantation Analysis of Cultured Sorted LSK Cells After ExVivo Pharmacological Manipulation Cell Harvest and Repopulation

Cells were harvested from the wells prior to transplantation bypipetting up and down several times before transferring to a fresh tube.Residual was then collected by adding more media and repeating. Cellswere washed in DMEM (Invitrogen, Cat. No. 31053) without phenol red andadded to the appropriate number of whole bone marrow rescue cells from acongenic donor (for 200,000 rescue cells+1,000 re-sorted LSK Flk2⁻ cells(FIGS. 3F-H) or the non-adherent product of 10 days culture of 100 LSKFlk2⁻ cultured cells (FIGS. 3I-K) per mouse as indicated). Cells wereinjected into lethally irradiated (10 Grays, single dose) Ptprc(CD45.1⁺) recipient mice through the tail vein using an insulin syringe.

Repopulation was measured at 4 weeks post-transplant by collection ofperiperal blood, red blood cell lysis, and staining of CD45.1(recipient) compared to CD45.2 (donor) engraftment using antibodiespurchased from eBiosciences (FITC conjugated CD45.2 (Cat. No.11-0454-85) and PE-Cy5 conjugated CD45.1 (Cat. No. 15-0453-82)). Micetransplanted with rescue/competitor cells only were used as a control todetermine the limits of repopulation detection. Multi-lineagereconstitution was determined by CD3, B220 (for lymphoid) and Gr1, Mac-1(for myeloid), as described above.

Transplantation Analysis of 28 Day Cultures.

Cells cultured for 28 days in (1) media, (2) media+BpV(pic), (3)media+CHIR99021 and (4) media+CHIR99021 (1 μM)+BpV(pic) (200 nM) werere-sorted for LSK Flk2⁻ cells. One thousand LSK Flk2⁻ cells (CD45.2⁺)from each media condition were transplanted into lethally irradiated(10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bonemarrow competitor cells. At 4 weeks post-transplant, peripheral bloodwas analyzed for donor (FIG. 3G) and multi-lineage (FIG. 3H)engraftment. In FIG. 3G, each bar represents an individual mouse. Thehorizontal-dashed line represents the average ‘engraftment’ of micetransplanted with competitor cells only and, thus, the limit ofdetectability for true engraftment. Long-term (4 month) engraftment hasnot been observed from 28-day cultures (data not shown). Six of 8 miceshow >1% engraftment when transplanted with LSK Flk2⁻ cells culturedwith both inhibitors present compared to 4/8 with only CHIR99021present, 0/10 with only BpV(pic) present, and 2/6 with media only. Onepercent or greater engraftment is a standard limit for substantialengraftment. (Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang,C. C. and H. F. Lodish, Blood, 105(11): 4314-20, 2005). Thus, while bothinhibitors together leads to greatest expansion in LSKs (FIG. 2F),transplantation of equivalent numbers of these cultured LSK Flk2⁻ cellsalso leads to increased short-term engraftment/functionality whencultured with both inhibitors compared to no or either single inhibitoronly.

While all mice with genetic alterations resulting in constitutivelyactive β-catenin and loss of PTEN will develop leukemia and must besacrificed due to poor health within 8-10 weeks post-mutation induction(FIG. 1I and data not shown), no mice transplanted with LSK Flk2⁻ cellscultured in either inhibitor singly or in combination has shown any signof leukogenesis up to 16 weeks post-transplantation. All such miceappeared healthy unlike 8-10 weeks post-induction genetically doublemutant mice, exhibiting no loss of body weight, anemia, loss ofappetite, lethargy, hunched posture, etc. Thus, the effects of theinhibition of both pathways using, e.g., BpV(pic) and CHIR99021, isreversible.

Transplantation Analysis of 10 Day Cultures.

Cells cultured for 9 days in (1) media, (2) media+BpV(pic) (200 nM), (3)media+CHIR99021 (100 nM), and (4) media+CHIR99021 (100 nM)+BpV(pic) (200nM) were re-sorted for LSK Flk2⁻ cells, and fold expansion of LSK Flk2⁻cells after 9 days culture in the indicated conditions was determined(FIG. 3I). Because long-term engraftment was not observed from 28 daycultures (FIGS. 3D-H and data not shown), LSK Flk2⁻ cells were culturedfor only 9 days to test if both expansion and long-term repopulationcould be achieved. Similar trends were observed here when compare to the28 day cultures (compare to FIG. 9F) although the extent of expansionwas substantially reduced at only 9 days versus 28 days culture.

FACS analysis was performed on 9 day LSK Flk2⁻ cells cultured inmedia+BpV(pic) (200 nM)+CHIR99021 (100 nM) (FIG. 3J). Cells werepre-gated on live, lineage negative cells. Greater than 90% of LSKsretain Flk2 negativity (data not shown). Here, the levels of Sca-1 andKit appear normal compared to the Sca-1^((high))Kit^((high)) populationshown from 28 day cultures (FIG. 1E).

Ten day cultures were transplanted into lethally irradiated (10Gy)CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bone marrowcompetitor cells. The total, non-adherent cell product after 10 daysculture of 100 initial LSK Flk-2 cells was transplanted per mouse. At 8weeks post-transplant, peripheral blood was analyzed for donor (FIG. 3G)and multi-lineage (FIG. 3H) engraftment. As shown, multi-lineagereconstitution was observed from all mice exhibiting true engraftment(data not shown). In FIG. 3G, each bar represents an individual mouse;the horizontal-dashed line represents the average ‘engraftment’ of micetransplanted with competitor cells only and thus the limit ofdetectability for true engraftment. Here, 3/7 mice transplanted with LSKFlk2⁻ cells cultured in the presence of both inhibitors exhibited 1% orgreater donor engraftment compared to no mice reaching this threshold inthe single or no inhibitor groups.

Collectively, these data demonstrate that the PTEN/Akt and Wnt/β-cateninsignaling pathways can be manipulated pharmacologically to drive HSCexpansion. Functional, short-term HSCs show highest reconstitutionability when cultured in the presence of both inhibitors. Substantiallonger-term reconstitution (8 weeks) occurs only when HSCs are culturedin the presence of both inhibitors but not when cultured with eithersingle inhibitor or in the absence of either inhibitor. Thus, thepharmacological manipulation of both pathways simultaneously results inthe greatest expansion of functional HSCs. This effect is reversiblebecause recipient animals did not develop leukemia as genetic mutantsdid (FIG. 1) and cultured HSCs were able to differentiate unlikecultured HSCs from genetic mutants (FIG. 2).

Example 9 Transplantation Analysis of HSCs in a Population of BoneMarrow Mononuclear Cells Materials and Methods

For the experiments set forth in this Example, a particular HSCexpansion media was used. This HSC expansion media consists of thefollowing ingredients: (1) StemSpan Media (Stem Cell Technologies; Cat.No. 09600) (StemSpan Media consists of Iscove's-modified Dulbecco'smedium (IMDM) supplemented with 1% bovine serum albumin, 10 μg/mlrecombinant human insulin, 200 μg/ml iron-saturated transferrin, 0.1 mM2-mercaptoethanol and 2 mM glutamine.); (2) 10 ug/ml Heparin (Sigma;Cat. No. H-3149); (3) 0.5× Penicillin/Streptomycin (Sigma; Cat. No.P4333); (4) 10 ng/ml recombinant mouse (rm) or recombinant human (rh)Stem Cell Factor (SCF) (Biovision; Cat. No. 4328-10 or 4327-10,respectively); (5) 20 ng/ml rm or rh Thrombopoietin (Tpo) (CellSciences, Inc; Cat. No. CRT401B or CRT400B, respectively). CHIR99021(250 nM) (Stemgent, Inc; Cat. No. 04-0004) may be added to this HSCexpansion media as indicated.

Cells were cultured in 96-well U-bottom tissue culture plates (Becton,Dickinson and Company; Cat. No. 353077).

Antibodies used are listed below and as set forth in Example 1. Thefollowing antibodies were obtained from eBiosciences: FITC conjugatedCD45.2 (Cat. No. 11-0454-85), PE-Cy5 conjugated CD45.1 (Cat. No.15-0453-82), PE conjugated CD34 (Cat. No. 12-0349-73), and APCconjugated CD38 (Cat. No. 17-0389-73).

Cell counts were obtained using a Quanta cell counter/cytometer(Beckman-Coulter). Cell sorting and analysis were performed using aMoFlo (Dako, Ft. Collins, Colo.) flow cytometer and/or a CyAn ADP (Dako,Ft. Collins, Colo.). Frequency of LSK Flk⁻ cells was determined byanalyzing >3×10⁵ cells per sample independently in triplicate.

Bone marrow cells were harvested from C57BI/6 (CD45.2) mice and madeinto a single cell suspension by gently drawing through a 22 g needleseveral times. Mobilized peripheral blood or bone marrow from humanpatients was harvested at the University of Kansas Medical Center(Kansas City, Mo. USA). Because red blood cell (RBC) lysis wasdetermined to severely inhibit functional HSC expansion, cells were notexposed to any RBC lysis procedure. Mononuclear cells were isolated frommouse bone marrow using Histopaque 1077 (Sigma; Cat. No. 10771) andhuman blood or bone marrow using Ficoll-Paque PLUS (Stem CellTechnologies; Cat. No. 07917) according to the manufacturers'instructions. Cells were washed and resuspended in HSC expansion media.Cells were counted and a fraction of mononuclear cells (MNCs) werestained for lineage markers using CD3, CD4, CD8, 8220, IgM, Mac-1, Gr1,and Ter119 antibodies along with Kit, Sca-1, and Flk2 for mouse HSCanalysis or CD34 and CD38 for human HSC analysis. 1×10⁶ cells/0.1 mlwere stained at 4° C. for 30 minutes using 0.05 μg of antibody for eachlineage marker and 0.2 μg for remaining antibodies. Cells were washedtwice in staining buffer (1× Phosphate buffered saline (PBS) (Mediatech,Inc, Cat. No. 20-031-CV)+2% fetal bovine serum (FBS) (Gibco-BRL, Cat.No. 16140-071)). Frequency of putative HSCs (lineage negative, Sca-1⁺,Kit⁺, Flk-1⁻ for mouse or CD34⁺, CD38⁻ cells for human) was determinedby analyzing >3×10⁵ cells per sample independently in triplicate. MNCswere then plated at 100 putative HSCs (along with 2.5-5.0×10⁴ MNCsdepending on frequency of putative HSCs in the particularsample—typically 0.2-0.4%) in 200 μl of HSC expansion media per well ina 96-well U bottom plate (Becton, Dickinson and Company; Cat. No.353077). MNCs were also plated at 50 LSK Flk2⁻ cells (along with1.7-5.0×10⁴ MNC cells depending on frequency of putative HSC in theparticular sample—typically 0.1-0.3%) in 200 μl of HSC expansion mediaper well in a 96-well U-bottom plate. Cells were incubated at 37° C.with 5% CO₂ and 5% O₂ (balance N₂) for 14 days. Cultures were checkeddaily and cell pellets accumulating at the bottom of each well whichexceeded 2 mm in diameter were split into new wells at a 1:1 ratio(splitting involved resuspension of the culture cell pellet by gentlypipetting up and down 5-7 times and removing ½ of the volume of theoriginal well and placing it into a fresh well. That volume of freshmedia was then replaced in each “old” and “new” well). It is criticalfor optimal HSC expansion that cell pellets are maintained at a densityof 1-2 mm in size. Splitting is typically required at day 1 and every2-3 days thereafter. In parallel, putative HSCs were sorted into 96-wellU bottom plates at 100 putative HSCs per well. Sorted putative HSCs werehandled equivalently to unsorted cultures. After 14 days culture, thetotal culture product was harvested by pipetting up and down 10 timesand combining into a test tube. Cells were washed and resuspended inDMEM (Invitrogen; Cat. No. 31053) in a volume equivalent to 5 originalinput putative HSCs per 100 μl for unsorted HSC cultures (for example, awell containing MNCs with 100 putative HSCs along with its descendantwells resulting from splitting would be resuspended in 2,000 μl) or 100original input putative HSCs per 100 μl for sorted HSC cultures. Forcompetitive repopulation assays, 1×10⁵ bone marrow cells congenic withthe host (CD45.1⁺) were included per mouse. 100 μl of cultured cells orcells freshly isolated and quantified in the same manner weretransplanted into lethally irradiated (10 Grays, single dose) Proteintyrosine phosphatase, receptor type, C (Ptprc or CD45.1) recipient micethrough the tail vein using an insulin syringe (29 gauge). Mice wereplaced on Batril® water (Bayer Healthcare, LLC, Shawnee Mission, Kans.)3 days prior to irradiation which continued for 2 weekspost-irradiation. Repopulation was measured every 4 weekspost-transplant by collection of periperal blood, red blood cell lysisand staining of CD45.1 (recipient) vs. CD45.2 (donor) engraftment. Micetransplanted with rescue/competitor cells only were used as a control todetermine the limits of detectable repopulation (typically 0.2%).Multi-lineage reconstitution was determined by CD3, 8220 (for T and Blymphoid, respectively) and Gr1, Mac-1 (for myeloid) gating on donor(CD45.2⁺) cells. For secondary transplantation, the original, primarytransplant recipients were sacrificed and bone marrow was harvested fromthe femur, made into a single-cell suspension, and strained through a 70μM cell strainer (BD Biosciences; Cat. No. 21008-952). Bone marrow cellswere counted and transplanted as above at a dosage of 1x10⁶ per mouse.

Experimental Results

The results show that ex vivo expansion of unsorted bone marrowmononuclear cells enhances functional long-term hematopoieticreconstitution relative to sorted, ex vivo expanded HSCs (FIG. 4). Thesedata demonstrate that the culture methodology set forth above results insubstantial expansion of functional HSCs with long-term, multi-lineagerepopulating potential. The presence of non-stem cells is critical tothis expansion, demonstrating that the typical practice of purifyingspecific putative HSC populations is not ideal for the ex vivo expansionof HSCs. Indeed, the cultured product of MNCs containing only 5 putativeHSCs exhibits increased repopulation potential compared to 100 sortedputative HSCs which are either freshly isolated or also cultured.Secondary transplant experiments further demonstrate that functional,long-term repopulating HSCs have been expanded with all recipientsexhibiting >25% donor repopulation with the average being >60%. Incontrast, an equivalent sample of unexpanded MNCs yields long-term (16+weeks) donor repopulation of <1% in all recipients, with ⅖ recipientsbeing at or below levels of detectable engraftment. This cultureexpansion protocol meets rigorous functional tests, including theability to yield high levels of repopulation even in the presence of 10⁵fresh, uncompromised competitor cells and in serial transplantationexperiments, conditions that are generally more rigorous than thoseencountered clinically.

The results further showed that culture with the small-moleculeinhibitor of GSK-3β, CHIR99021, enhances long-term engraftment of exvivo expanded HSCs (FIGS. 5 and 13). While 100 sorted, putative HSCscultured without CHIR99021 yield average repopulation of 1.1%, culturingwith CHIR99021 yields average repopulation of 12.3%. Similarly, unsortedMNC cultures in the absence and presence of CHIR99021 yields averagerepopulation of 37.4 and 64.8%, respectively. These data demonstratethat ex vivo expansion in the presence of a small molecule inhibitor ofGSK-3β, CHIR99021, substantially increases the level of long-term,multi-lineage engraftment.

Although more mature, radioprotective cells are necessary for short-termsurvival of mice transplanted with enriched, putative HSCs (Na Nakorn,T., Traver, D., Weissman, I. L. & Akashi, K. Myeloerythroid-restrictedprogenitors are sufficient to confer radioprotection and provide themajority of day 8 CFU-S. The Journal of clinical investigation 109,1579-1585 (2002)), the inventors hypothesized that the culture systemmight allow for the transplantation of only ex vivo expanded cellswithout any competitor/radioprotective bone marrow cells. This wouldmake the culture system more relevant to and useful in a potentialclinical setting. To test this, after 14 days culture in ST media withand without CHIR99021, only the cultured product of MNCs containing 5LSK Flk2⁻ cells were transplanted into lethally irradiated recipients.For comparison, fresh, uncultured MNCs containing 5 LSK Flk2⁻ cells permouse were also transplanted. In addition, because the average 14-dayexpansion of LSK Flk2⁻ cells in the unsorted cultures was approximately100-fold (data not shown), fresh, uncultured MNCs containing 500 LSKFlk2⁻ cells were also transplanted into lethally irradiated recipientsfor comparison. While recipients of uncultured MNCs containing 5 LSKFlk2⁻ cells had to be sacrificed due to bone marrow failure between 2-3weeks post-irradiation, mice transplanted with only cultured cellscontaining 5 LSK Flk2⁻ cells or fresh MNCs containing 500 LSK Flk2⁻cells recovered. In these surviving groups, all primary recipientsexhibited robust donor engraftment (>90%) with no significant differencebetween groups (data not shown). To determine if CHIR99021 was affectingthe long-term potential of HSCs, the primary recipients and performedserial bone marrow transplantation into secondary, lethally-irradiatedrecipients. At 16 weeks post-secondary transplant, donor reconstitutionwas 67.3±20.6% for ex vivo expansion in ST media and 90.6±4.8% in STmedia with CHIR99021 (FIGS. 6G and 6H). Notably, there was nosignificant difference in long-term, multi-lineage donor reconstitutionbetween mice receiving ex vivo expanded HSCs in the presence ofCHIR99021 and mice receiving a 100-fold greater dosage of fresh LSKFlk2⁻ cells (90.6±4.8% vs. 90.1±3.1, respectively; p=0.88). These datademonstrate that functional LT-HSCs can be expanded ex vivo to asignificantly greater degree when they are not fractionated from moremature cells. Furthermore, by manipulating the Wnt/β-catenin pathways,inhibition of GSK3β with CHIR99021 during ex vivo HSC expansionsubstantially enhances long-term donor reconstitution.

These data demonstrate that the ex vivo expansion protocol allows fortransplantation of only the cultured product of MNCs containing 5putative HSCs, resulting in long-term survival of recipients. No fresh,rescue bone marrow cells are required. In contrast, transplantation offresh, unexpanded MNCs containing 5 putative HSCs does not allow any ofthe recipients to survive beyond 2-3 weeks, the typical survival time ofmice receiving lethal irradiation without transplantation (Na Nakorn,T., Traver, D., Weissman, I. L. & Akashi, K. Myeloerythroid-restrictedprogenitors are sufficient to confer radioprotection and provide themajority of day 8 CFU-S. The Journal of clinical investigation 109,1579-1585 (2002)). Thus, in addition to the expansion of long-termrepopulating HSCs, short-term radioprotective cells are also expandedutilizing the ex vivo expansion protocol. With the inclusion ofCHIR99021 during ex vivo expansion, the level of repopulation ofrecipients of ex vivo expanded MNCs containing 5 putative HSCs isequivalent to fresh, unexpanded MNCs containing 500 putative HSCs at 16weeks post-secondary transplantation. This data demonstrates that the exvivo expansion protocol allows for long-term repopulation equivalent toa 100-fold greater dose of fresh, unexpanded cells.

The data obtained from experiments involving ex vivo expansion of humanHSCs (FIG. 7) indicate that the culture methodology developed in themouse system should translate into the human system, allowing forsubstantial expansion of HSCs in culture. This should allow forcurrently limited sources of HSCs, such as umbilical cord blood, to beutilized with greater efficacy.

HSCs are known to be able to undergo considerable expansion in Vivo andare the most extensively studied stem cell system. It is somewhatparadoxical, therefore, that they remain difficult to culture, with onlymodest expansion being consistently achieved, while more significantexpansion is coupled with substantial differentiation (North, T. E. etal. Prostaglandin E2 regulates vertebrate haematopoietic stem cellhomeostasis. Nature 447, 1007-1011 (2007).; Kobayashi, M., Laver, J. H.,Kato, T., Miyazaki, H. & Ogawa, M. Thrombopoietin supports proliferationof human primitive hematopoietic cells in synergy with steel factorand/or interleukin-3. Blood 88, 429-436 (1996); Antonchuk, J.,Sauvageau, G. & Humphries, R. K. HOXB4-induced expansion of adulthematopoietic stem cells ex vivo. Cell 109, 39-45 (2002); Varnum-Finney,B. et al. Pluripotent, cytokine-dependent, hematopoietic stem cells areimmortalized by constitutive Notch1 signaling. Nat Med 6, 1278-1281(2000)). Driving self-renewal appears to require activation of certainproto-oncogenes along with simultaneous inhibition of certain tumorsuppressors, a combination that limits regenerative capacity and makessubstantial expansion difficult without risking oncogenesis or stem cellexhaustion (Reya, T. et al. A role for Wnt signaling in self-renewal ofhaematopoietic stem cells. Nature 423, 409-414 (2003); Yilmaz, O. H. etal. Pten dependence distinguishes haematopoietic stem cells fromleukaemia-initiating cells. Nature 441, 475-482 (2006); Zhang, J. et al.PTEN maintains haematopoietic stem cells and acts in lineage choice andleukaemia prevention. Nature 441, 518-522 (2006); Varnum-Finney, B. etal. Pluripotent, cytokine-dependent, hematopoietic stem cells areimmortalized by constitutive Notch1 signaling. Nat Med 6, 1278-1281(2000); Matsuoka, S. et al. Fbxw7 acts as a critical fail-safe againstpremature loss of hematopoietic stem cells and development of T-ALL.Genes Dev 22, 986-991 (2008); Park, I. K. et al. Bmi-1 is required formaintenance of adult self-renewing haematopoietic stem cells. Nature423, 302-305 (2003); Perry, J. M. & Li, L. Self-renewal versustransformation: Fbxw7 deletion leads to stem cell activation andleukemogenesis. Genes Dev. 22, 1107-1109 (2008)). Simultaneousmanipulation of proto-oncogene and tumor suppressor activity can achievesubstantial stem cell expansion in vitro; however, it is critical tobalance this transient expansion with return to conditions that mimicthe in vivo situation where relative quiescence is recovered and tumorsuppressors are reactivated.

Although sorting specific populations enriched in HSCs has been thetypical methodology utilized for culturing HSCs, the inventors found exvivo HSC expansion to be best supported when cultured in the presence ofmore mature cells. Indeed, when LSK Flk2⁻ cells were sorted, substantialHSC expansion was achieved only after some differentiation had occurred.It may be that HSCs negatively inhibit self-renewal and even survival ofother HSCs in close proximity, helping to maintain stem cells as a rarepopulation in vivo. Culturing unsorted HSCs combined with othertechnical procedures results in robust functional HSC expansion. Whilethe ex vivo HSC expansion protocol yielded robust long-termreconstitution, even in competitive repopulation assays, competitor orrescue bone marrow cells were not necessary following ex vivo expansion,demonstrating that radioprotective cells also expanded in the culturesystem. Utilizing a small molecule inhibitor of GSK3β, the ex vivo HSCexpansion protocol allowed for expansion of LT-HSCs which performedequivalently to a 100-fold greater dosage of uncultured cells. Employinga defined culture media with relatively low concentrations of only twocytokines, but without the necessity of feeder layers, cell sorting, orthe use of fresh bone marrow for radioprotection, this culture systemmay have clinical value if developed for humans.

In summary, this process for the ex vivo expansion of hematopoietic stemcells utilizes defined culture media supplemented with lowconcentrations of only two specific cytokines and does not requirecomplicated schemes such as cell sorting or contaminating cellularfeeder layers. Therefore, it allows for fast, simple and relativelyinexpensive expansion of functional HSCs. In addition, HSCtransplantation following myeloablative therapy requires thetransplantation of radioprotective cells, typically whole bone marrowcells, for short-term survival prior to the establishment of long-termhematopoiesis by HSCs (Paquette, R. & Dorshkind, K. Optimizinghematopoietic recovery following bone marrow transplantation. TheJournal of clinical investigation 109, 1527-1528 (2002)). This HSCexpansion protocol also expands these radioprotective cells, allowingfor the transplantation of the cultured product alone. When culturedwith the small molecule CHIR99021, an inhibitor of GSK-3β, this ex vivoexpansion protocol allows for long-term repopulation equivalent to a100-fold greater dose of fresh, unexpanded cells.

Example 10 Culturing of HSC in Media Containing Biologics

Anti-GSK-3β and anti-PTEN antibodies may be made in accordance withprocedures known in the art (or purchased, e.g., from Sigma,ExactAntigene, and Biocompare).

One hundred LSK Flk2⁻ cells are sorted from wild-type (C57BI/6) mice andare cultured in (1) media, (2) media+an GSK-3β antibody, (3) media+ananti-PTEN antibody, and (4) media+anti-GSK-3β and anti-PTEN antibodies.Cells are cultured as described above. Cells are examined at 9 days, 17days and 23 days of culture. The greatest expansion of HSCs is expectedto occur when both antibodies are present.

Example 11 Culturing of HSC in Media Containing siRNA or RNAi

PTEN siRNA and GSK-3b siRNA may be made in accordance with proceduresknown in the art. (See, e.g., Mise-Omata S et al. Biochem Biophys ResCommun. 328(4):1034-42 2005, or may be purchased from Biocompare).

One hundred LSK Flk2⁻ cells are sorted from wild-type (C57BI/6) mice andare cultured in (1) media, (2) media+GSK-3β siRNA, (3) media+PTEN siRNA,and (4) media+GSK-3β siRNA and PTEN siRNA. Cells are cultured asdescribed above. Cells are examined at 9 days, 17 days and 23 days ofculture. The greatest expansion of HSC is expected to occur when bothsiRNAs are present.

All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

1. An ex vivo method for expanding the number of hematopoietic stemcells (HSC) in a population of mononuclear cells (MNC) comprisingculturing the population of MNCs comprising at least one HSC in an HSCexpansion media for a period of time sufficient to expand the number ofHSCs in the MNC population, wherein the expanded HSCs are functionalwith long term, multi-lineage, repopulating potential.
 2. The methodaccording to claim 1, which provides HSCs that, upon transplant into arecipient, exhibit greater than 5% donor repopulation.
 3. The methodaccording to claim 1, which provides HSCs that, upon transplant into arecipient, exhibit greater than 25% donor repopulation.
 4. The methodaccording to claim 1, which provides HSCs that, upon transplant into arecipient, exhibit greater than 45% donor repopulation.
 5. The methodaccording to claim 1, which provides HSCs that, upon transplant into arecipient, exhibit greater than 60% donor repopulation.
 6. The methodaccording to claim 1, wherein the HSC expansion media comprises amodulator of the Wnt pathway.
 7. The method according to claim 6,wherein the modulator of the Wnt pathway down-regulates GSK-3β.
 8. Themethod according to claim 6, wherein the modulator of the Wnt pathway isa reversible GSK-3β inhibitor selected from the group consisting of asmall molecule, a biologic, an antisense RNA, a small interfering RNA(siRNA), and combinations thereof.
 9. The method according to claim 8,wherein the reversible GSK-3β inhibitor is a small molecule.
 10. Themethod according to claim 8, wherein the reversible GSK-3β inhibitor isselected from the group consisting of Hymenialdisine, Flavopiridol,Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, AloisineA, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021),CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a,Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220(bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474, Compound 8b,TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, smallmolecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12(Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-LaRoche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, Universityof Rochester), pharmaceutically acceptable salts thereof, andcombinations thereof.
 11. The method according to claim 10, wherein theGSK-3β inhibitor is CHIR99201.
 12. The method according to claim 6,which provides HSCs that, upon transplant into a recipient, exhibitgreater than 60% donor repopulation.
 13. The method according to claim1, wherein the HSC is obtained from a mammalian tissue selected from thegroup consisting of cord blood, peripheral blood, and bone marrow. 14.An expanded, substantially undifferentiated HSC population made by themethod according to claim
 1. 15. An expanded, substantiallyundifferentiated HSC population made by the method according to claim 6.16. A kit for expanding, ex vivo, the number of hematopoietic stem cells(HSC) in a population of mononuclear cells (MNC), the kit comprising aGSK-3β inhibitor, and instructions for the use of the inhibitor,wherein, when used, the kit provides expanded HSCs that are functionalwith long term, multi-lineage, repopulating potential.
 17. The kitaccording to claim 16, wherein the GSK-3β inhibitor is selected from thegroup consisting of Hymenialdisine, Flavopiridol, Kenpaullone,Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, AloisineA, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021),CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a,Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220(bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474, Compound 8b,TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, smallmolecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12(Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-LaRoche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, Universityof Rochester), pharmaceutically acceptable salts thereof, andcombinations thereof.
 18. The kit according to claim 16, wherein theGSK-3β inhibitor is CHIR99201.
 19. The kit according to claim 16, whichprovides HSCs that, upon transplant into a recipient, exhibit greaterthan 60% donor repopulation.
 20. A media for carrying out ex vivoexpansion of a stem cell in a population of MNCs comprising a fluidmedia suitable for maintaining viable stem cells and a GSK-3β inhibitorpresent in the media at a concentration sufficient to enable expansionof the stem cell population while maintaining a long term,multi-lineage, repopulating potential in the stem cells, wherein thestem cells, when transplanted into a recipient, exhibit greater than 5%donor repopulation.
 21. An ex vivo method for expanding the number ofcells capable of supporting multi-lineage repopulation in a populationof mononuclear cells (MNC) comprising culturing the population of MNCscomprising at least one hematopoietic stem cell (HSC) and at least onehematopoietic progenitor cell in an HSC expansion media for a period oftime sufficient to expand the number of cells capable of supportingmulti-lineage repopulation in the MNC population.